Melanocortin 5 receptor signaling pathways: implications on adipocyte biology.

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(1)MELANOCORTIN 5 RECEPTOR SIGNALING PATHWAYS: IMPLICATIONS ON ADIPOCYTE BIOLOGY ADRIANA RAQUEL CAMPOS RODRIGUES. TESE DE DOUTORAMENTO APRESENTADA À FACULDADE DE MEDICINA DA UNIVERSIDADE DO PORTO EM BIOMEDICINA.

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(3) TESE DE DOUTORAMENTO APRESENTADA À FACULDADE DE MEDICINA DA UNIVERSIDADE DO PORTO NO ÂMBITO DO PROGRAMA DOUTORAL EM BIOMEDICINA. Orientação da Professora Doutora Alexandra Maria Monteiro Gouveia Co-orientação do Professor Doutor Henrique Manuel Nunes de Almeida. MELANOCORTIN 5 RECEPTOR SIGNALING PATHWAYS: IMPLICATIONS ON ADIPOCYTE BIOLOGY MECANISMOS MOLECULARES DE SINALIZAÇÃO DO RECETOR 5 DAS MELANOCORTINAS E SUAS IMPLICAÇÕES NA BIOLOGIA DO ADIPÓCITO ADRIANA RAQUEL CAMPOS RODRIGUES. PORTO, 2013.

(4) Artigo 48º, § 3º. “A Faculdade não responde pelas doutrinas expendidas na dissertação”. (Regulamento da Faculdade de Medicina do Porto, Decreto-Lei nº 19337 de 29 de Janeiro de 1931).

(5) CORPO CATEDRÁTICO DA FACULDADE DE MEDICINA DO PORTO Professores Efetivos. Alberto Manuel Barros da Silva Altamiro Manuel Rodrigues Costa Pereira Álvaro Jerónimo Leal Machado de Aguiar António Carlos Freitas Ribeiro Saraiva Daniel Filipe Lima Moura Deolinda Maria Valente Alves Lima Teixeira Francisco Fernando Rocha Gonçalves Isabel Maria Amorim Pereira Ramos João Francisco Montenegro Andrade Lima Bernardes Jorge Manuel Mergulhão Castro Tavares José Agostinho Marques Lopes José Carlos Neves da Cunha Areias José Eduardo Torres Eckenroth Guimarães José Henrique Dias Pinto de Barros José Manuel Lopes Teixeira Amarante José Manuel Pereira Dias de Castro Lopes Manuel Alberto Coimbra Sobrinho Simões Manuel António Caldeira Pais Clemente Manuel Jesus Falcão Pestana Vasconcelos Maria Amélia Duarte Ferreira Maria Dulce Cordeiro Madeira Maria Fátima Machado Henriques Carneiro Maria Leonor Martins Soares David Patrício Manuel Vieira Araújo Soares Silva Rui Manuel Almeida Mota Cardoso Rui Manuel Lopes Nunes. Professores Jubilados ou Aposentados. Abel José Sampaio da Costa Tavares Abel Vitorino Trigo Cabral Alexandre Alberto Guerra Sousa Pinto Amândio Gomes Sampaio Tavares António Augusto Lopes Vaz António Carvalho Almeida Coimbra António Fernandes da Fonseca António Fernandes Oliveira Barbosa Ribeiro Braga António Germano Pina Silva Leal António José Pacheco Palha António Luís Tomé da Rocha Ribeiro António Manuel Sampaio de Araújo Teixeira Belmiro dos Santos Patrício Cândido Alves Hipólito Reis Carlos Rodrigo Magalhães Ramalhão Cassiano Pena de Abreu e Lima Daniel Santos Pinto Serrão Eduardo Jorge Cunha Rodrigues Pereira Fernando de Carvalho Cerqueira Magro Ferreira Fernando Tavarela Veloso Francisco de Sousa Lé Henrique José Ferreira Gonçalves Lecour de Menezes Joaquim Germano Pinto Machado Correia da Silva José Augusto Fleming Torrinha José Carvalho de Oliveira José Fernando Barros Castro Correia José Luís Medina Vieira José Manuel Costa Mesquita Guimarães Levi Eugénio Ribeiro Guerra Luís Alberto Martins Gomes de Almeida Manuel Augusto Cardoso de Oliveira Manuel Machado Rodrigues Gomes Manuel Maria Paula Barbosa Maria da Conceição Fernandes Marques Magalhães Maria Isabel Amorim de Azevedo Mário José Cerqueira Gomes Braga Serafim Correia Pinto Guimarães Valdemar Miguel Botelho dos Santos Cardoso Walter Friedrich Alfred Osswald.

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(7) JURÍ NOMEADO PARA A PROVA DE DOUTORAMENTO Presidente:. Reitor da Universidade do Porto. Vogais:. Doutora Lídia Mariana Rodrigues Pereira Monteiro, professora associada do Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto;. Doutor Davide Maurício Costa Carvalho, professor associado da Faculdade de Medicina da Universidade do Porto;. Doutora Delminda Rosa Gamelas Neves Lopes de Magalhães, professora associada da Faculdade de Medicina da Universidade do Porto;. Doutora Cláudia Margarida Gonçalves Cavadas, professora auxiliar da Faculdade de Farmácia da Universidade de Coimbra; Doutora Alexandra Maria Monteiro Gouveia, professora auxiliar convidada da Faculdade de Medicina da Universidade do Porto e orientadora da tese; Doutor Peter Jordan, investigador principal do Instituto Nacional de Saúde Doutor Ricardo Jorge..

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(9) À Professora Doutora Alexandra Gouveia. Ao Professor Doutor Henrique Almeida.

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(11) Aos meus pais e ao meu irmão. Ao Pedro.

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(13) P. REFÁCIO. Foi de facto uma bênção ter tido a oportunidade de desenvolver o meu trabalho de doutoramento no Departamento de Biologia Experimental da Faculdade de Medicina do Porto. Nem sempre foi assim conhecido, no meu primeiro contacto com a ciência, há já alguns anos, ele era chamado de Instituto de Histologia e Embriologia e tinha morada num corredor estreito com laboratórios e gabinetes à mistura. Muito mudou desde esse tempo! Mas o ambiente acolhedor e as pessoas fantásticas que conheci nessa época desenvolveram em mim a secreta vontade de lá continuar… e assim tem sido! Esse privilégio, eu devo-o em especial à minha orientadora, a Professora Doutora Alexandra Gouveia, a quem expresso o meu profundo reconhecimento quer pela extraordinária capacidade científica quer pelo anseio contagiante e entusiasmante de procurar saber sempre mais. Os trabalhos apresentados nesta dissertação simbolizam a dedicação, o esforço e o rigor metódico de uma orientação admirável, com a qual podia contar todos os dias, a toda a hora. Para além dos ensinamentos científicos que muito me fizeram crescer enquanto investigadora, eu agradeço-lhe sobretudo a amizade e humanidade características da sua forma de ser. Obrigada por teres sido muito mais que uma orientadora inestimável, por teres sido uma amiga e uma conselheira! Ao Professor Doutor Henrique Almeida, meu co-orientador, expresso a minha admiração pela sua disponibilidade e empenho permanentes. Estou-lhe profundamente grata pela forma como me acolheu aquando do meu ingresso neste mundo da ciência e pela forma como me acompanhou ao longo destes anos promovendo e partilhando o meu desenvolvimento científico. À Professora Doutora Deolinda Lima, uma fonte de inspiração para qualquer cientista, agradeço a honra concedida ao abrir-me as portas da sua instituição, a qual dirige de forma notável.. À Professora Doutora Delminda Neves devo expressar o meu reconhecimento pela delicadeza e sensibilidade com que sempre me atendeu e agradecer o seu apoio e contributo de que muito beneficiou este trabalho. Às minhas colegas, companheiras e amigas, Liliana Matos e Inês Tomada, estou particularmente grata por toda a ajuda e pelo carinho sempre demonstrado. Tive o privilégio de com elas partilhar críticas e sugestões científicas por entre muitas alegrias e gargalhadas. Foram também muitas as melancolias e inquietações depois de inúmeras experiências mal sucedidas, mas a excelente cumplicidade e o apoio assíduo com que nos deleitávamos todos os dias traziam o ânimo e a perseverança necessárias para que tudo fizesse sentido. Ao José Pedro Castro agradeço-.

(14) lhe a boa disposição que contagia todos os que perto dele trabalham. À Elizabete Silva e à Ana Isabel agradeço o dinamismo que trouxeram ao grupo e que muito o valorizou.. Ao Carlos Reguenga, desejo expressar o mais sincero e especial agradecimento por todos os ensinamentos técnicos e científicos que prontamente prestava e que desde cedo influenciaram a minha forma de fazer ciência. A sua disponibilidade constante, as suas críticas e sugestões, reveladores de um profundo conhecimento, e o seu excelente sentido de humor, que muitas vezes transforma uma reflexão científica numa estimulante troca de ideias, torna-o de facto uma mais-valia para qualquer laboratório de investigação. Ao Filipe Monteiro quero prestar o meu reconhecimento por toda a ajuda sempre pronta. Agradeço também à Mariana Matos e à Isabel Regadas pela ternura com que sempre se disponibilizaram a ajudar. Ao Ricardo Reis pelas enriquecedoras conversas e discussões científicas que fomos partilhando.. À Célia Cruz e ao Jorge Ferreira devo expressar a minha gratidão pela amizade e apoio que sempre demonstraram. A eles devo o meu entusiasmo pelo estudo das vias de sinalização celular que me persegue desde o meu estágio de licenciatura. À Carla Morgado, o meu obrigada pelo carinho, a presença amiga e a forma singela com que se preocupa. À Isabel Martins, com quem tive, em tempos, o privilégio de partilhar gabinete, agradeço a amizade, as sugestões e os concelhos. Também à Ana Charrua e à Fani Neto agradeço-lhes pelo excelente convívio e por serem ambas, ainda que de formas muito diferentes, tão generosas e genuínas. À Joana Gomes e à Clara Monteiro, parceiras desta última fase da escrita, estou grata pela troca de desabafos e palavras positivas que nos reforçava o desejo mútuo de chegar ao fim. Também ao Vasco Galhardo agradeço esse incentivo. Ao Professor António Avelino gostaria de agradecer as “valsas improvisadas” que muito desconcertadamente tentavam atenuar-me a ansiedade característica destas etapas finais. A todos os técnicos, nomeadamente à Elisa Nova e à Anabela Silvestre, estou grata pela colaboração e dedicação que imprimem na gestão do laboratório e à Raquel Madanços e Ana Tavares agradeço todo o apoio no trabalho de secretariado. A todos os demais colaboradores e investigadores do Departamento de Biologia, expresso o meu sincero agradecimento por toda a ajuda e companheirismo, contribuindo de forma exemplar para um ambiente fantástico e verdadeiramente acolhedor.. Aos meus pais devo toda a minha gratidão, inspiração e motivação. Agradeço-lhes o apoio e amor incondicionais. São e sempre foram os pilares da minha vida e o maior orgulho que trago no coração. Obrigada por fazerem de mim a pessoa que hoje sou. Ao meu irmão agradeço-lhe o afeto, a confiança e também a paciência extrema que sabia ter quando eu dela precisava. A sua inocência e jovialidade sempre tornaram fáceis os momentos mais difíceis. E talvez por isso, a ti devo muitas das melhores coisas que me aconteceram na vida. E finalmente, desejo agradecer ao Pedro o carinho, a cumplicidade e a dedicação imensuráveis. Obrigada pela presença e apoio constantes em todos os momentos, nos melhores e nos piores. A forma apaziguadora com que tão bem lidas com o meu maufeitio, típico de quando as coisas não me correm bem, é de facto heroica. E a forma como compensas esses dias menos bons continua a ser notável e apaixonante. Obrigada simplesmente por existires..

(15) Em obediência ao disposto no Decreto-Lei nº 388/70, Artigo 8º, parágrafo 2, declaro que efetuei o planeamento e execução das experiências, observação do material e análise de resultados e participei ativamente na redação de todas as publicações que fazem parte integrante desta dissertação: I. Rodrigues AR, Pignatelli D, Almeida H, Gouveia AM (2009) Melanocortin 5 receptor activates ERK1/2 through a PI3K-regulated signaling mechanism. Mol Cell Endocrinol, 303(1-2):74-81. II. Rodrigues AR, Almeida H, Gouveia AM (2012) Melanocortin 5 Receptor signaling and internalization: role of MAPK/ERK pathway and β-arrestins1/2. Mol Cell Endocrinol, 361(1-2):69-79.. III. Rodrigues AR, Almeida H, Gouveia AM (2013) α-MSH signaling via Melanocortin 5 Receptor promotes lipolysis and impairs re-esterification in adipocytes. Biochim Biophys Acta, 1831(7):1267-1275.. O trabalho apresentado nesta dissertação foi realizado no âmbito de uma bolsa de doutoramento (SFRH/BD/41024/2007) concedida pela Fundação para a Ciência e a Tecnologia..

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(17) RESUMO A obesidade é atualmente uma epidemia mundial e uma das maiores ameaças para. a saúde humana. Na obesidade, a acumulação excessiva de gordura resulta de um. desequilíbrio entre a ingestão alimentar e o gasto energético, dois mecanismos altamente regulados pelo sistema das melanocortinas. No sistema nervoso central, os recetores das. melanocortinas MC3R e MC4R ativam sinais de saciedade inibindo a ingestão alimentar, no entanto, o mecanismo através do qual as melanocortinas regulam o metabolismo lipídico. nos adipócitos é pouco conhecido. Considerando o envolvimento do recetor 5 das. melanocortinas (MC5R) no metabolismo lipídico noutros tecidos periféricos, este recetor surge como um potencial candidato na regulação da função do adipócito. Neste âmbito, esta tese tem como objetivo fornecer uma caracterização detalhada dos mecanismos de sinalização e tráfego intracelulares mediados pela ligação de melanocortinas ao MC5R, destacando o seu papel na biologia do adipócito.. As vias de sinalização mediadas pela ativação específica do MC5R foram estudadas. num sistema celular heterólogo com sobrexpressão estável do MC5R-GFP. A ligação da α-. MSH ao MC5R ativa duas vias de sinalização paralelas, a via adenosina monofosfato cíclico/proteína cinase A (cAMP/PKA) e a via das cinases reguladas por sinais extracelulares 1/2 (ERK1/2), cuja fosforilação ocorre independentemente da PKA, da. proteína cinase C (PKC) e da Akt, mas requer a ativação do fosfoinositol 3-cinase (PI3K).. Uma análise mais detalhada dos mecanismos moleculares envolvidos na sinalização e tráfego do MC5R demonstram que a via ERK1/2 é ativada de forma bifásica, com uma. ativação transitória inicial dependente da proteína Gi e uma fosforilação sustentada tardia. regulada pelas β-arrestinas. Embora pareçam funcionar como proteínas adaptadoras para. prolongar a sinalização das ERK1/2 e impedir a sua translocação nuclear, as β-arrestinas. não estão envolvidas na internalização do MC5R. Este processo ocorre via vesículas revestidas de clatrina e de seguida o MC5R é reciclado novamente para a membrana. No.

(18) entanto, o pico inicial da sinalização das ERK1/2, que depende da proteína Gi, é essencial para a translocação destas cinases para o núcleo, onde são capazes de induzir a expressão do fator de transcrição c-Fos. A ativação da cAMP/PKA pelo MC5R é dependente da proteína Gs e também promove a fosforilação de um segundo fator de transcrição, a proteína de ligação ao elemento de resposta ao cAMP (CREB).. A significância funcional destes mecanismos de sinalização foi posteriormente. analisada em adipócitos 3T3-L1 após silenciamento da expressão do MC5R por siRNA. Os nossos estudos demonstraram, pela primeira vez, que a ativação do MC5R em adipócitos. aumenta a lipólise e impede a re-esterificação de ácidos gordos por gliceroneogénese. O efeito lipolítico é mediado pela via cAMP/PKA e envolve a ativação da lipase hormona-. sensível (HSL), da lipase de triglicerídeos do adipócito (ATGL) e das perilipinas (PLIN), enquanto que a inibição da re-esterificação de ácidos gordos depende das ERK1/2 que bloqueiam a atividade da fosfoenolpiruvato carboxicinase (PEPCK).. No seu conjunto, os estudos que integram esta dissertação apontam claramente. para um papel fundamental do MC5R no metabolismo lipídico nos adipócitos: promove a. lipólise através da cAMP/PKA que depende da proteína Gs, e impede a re-esterificação de. ácidos gordos através da sinalização das ERK1/2, a qual é ativada por mecanismos. dependentes da proteína Gi e das β-arrestinas. É nossa convicção de que o conhecimento. desta modulação distinta da sinalização e função do MC5R possa ser útil para o futuro desenvolvimento de novas drogas, visando a especificidade, segurança e eficácia nos tratamentos de obesidade..

(19) ABSTRACT Obesity is currently a global pandemic and one of the biggest threats to human. health all over the world. The excessive fat accumulation during obesity results from an imbalance between food intake and energy expenditure, two mechanisms highly regulated by the melanocortin system. In the central nervous system, the melanocortin receptors. MC3R and MC4R activate satiety signals to inhibit feeding whereas the mechanism. through which melanocortins modulate lipid metabolism in adipocytes has been poorly investigated. Nevertheless, the melanocortin 5 receptor (MC5R) is one possible key player. in this biological process. In this setting, this thesis aims to provide a detailed characterization of the intracellular signaling and trafficking mechanisms mediated by the melanocortin binding to MC5R, highlighting its function on the adipocyte.. Through the development of an heterologous cell system stably expressing MC5R-. GFP, we were able to unravel the specific signaling pathways mediated by MC5R activation. MC5R elicits two parallel signals when activated by α-MSH: the cyclic adenosine. monophosphate/protein kinase A (cAMP/PKA) pathway and the extracellular signalregulated kinase 1/2 (ERK1/2) pathway, which phosphorylation occurs independently of. PKA, protein kinase C (PKC) and Akt but requires the phosphoinositide 3-kinase (PI3K). A. more detailed analysis of the molecular mechanisms conveyed by MC5R signaling and trafficking led us to find out that ERK1/2 pathway is activated in a biphasic fashion with. an early transient activation dependent from Gi protein and a late sustained. phosphorylation regulated by β-arrestins. Although they appear to function as scaffolds to. prolong ERK1/2 signaling and prevent their nuclear translocation, β-arrestins are not involved in MC5R internalization, which occurs through clathrin-coated vesicles and is. followed by recycling of the receptor to the cell surface. Nevertheless, the earlier Gi. protein dependent peak of ERK1/2 signaling is important for the translocation of these. kinases to the nuclei, where they are able to induce the expression of the transcription.

(20) factor c-Fos. The cAMP/PKA activation by MC5R is dependent from Gs protein and also. promotes the phosphorylation of a second transcription factor, the cAMP-response element binding protein (CREB).. The functional significance of cAMP/PKA and ERK1/2 pathways was further. ascertained in the adipocyte cell model 3T3-L1 after silencing MC5R expression by siRNA. techniques. Our studies demonstrated, for the first time, that MC5R activation in adipocytes. promotes. lipolysis. and. impairs. fatty. acids. re-esterification. by. glyceroneogenesis. The lipolytic effect is mediated by cAMP/PKA pathway and involves. the activation of the hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipins (PLIN) whereas the blockage on fatty acids re-esterification occurs through an ERK1/2-dependent inhibition of phosphoenolpyruvate carboxykinase (PEPCK) activity.. Altogether, the studies that integrate this dissertation clearly uncover a key role. for MC5R in the adipocyte lipid metabolism: it promotes lipolysis by a cAMP/PKA pathway depending on Gs protein, and impairs fatty acids re-esterification through ERK1/2 signaling which is driven by both Gi protein and β-arrestins-dependent mechanisms. We are convinced that the knowledge of this distinct modulation of MC5R signaling and. function may be helpful for the future designing of novel drugs targeting specificity, safety and effectiveness in obesity therapies..

(21) ABBREVIATIONS AC ACC ACTH AgRP AMPK AP-2 ARC ATGL cAMP CCPs CNS CREB DAG ER ERK1/2 FA FGD GEF GFP GPCR Grb2 GRK HSL IP3 JAK/STAT JNK MAPK MCR MGL MRAP MSK NEFA PC PEPCK PI3K PIP 2 PKA PKC PLCβ PLIN POMC RSK RTK Shc SNS SOS TG WAT β-AR α-MSH. Adenylyl cyclase Acetyl CoA carboxylase Adrenocorticotropic hormone Agouti-related protein Adenosine monophosphate-activated protein kinase Adaptor protein-2 Arcuate nucleus Adipose triglyceride lipase Cyclic adenosine monophosphate Clathrin-coated pits Central nervous system cAMP-response element binding protein Diacylglycerol Endoplasmic reticulum Extracellular signal-regulated kinase 1/2 Fatty acids Familial glucocorticoid deficiency Guanine exchange factor Green fluorescent protein G protein-coupled receptor Growth factor receptor-bound protein 2 GPCR kinase Hormone sensitive lipase Inositol 1,4,5 triphosphate Janus protein kinase/signal transducers and activators of transcription c-Jun N-terminal kinases Mitogen activated protein kinase Melanocortin receptor Monoacylglycerol lipase Melanocortin 2 receptor accessory protein Mitogen- and stress-activated protein kinase Non-esterified fatty acids Prohormone-convertase Phosphoenolpyruvate carboxykinase Phosphoinositide 3-kinase Phosphatidylinositol 4,5-bisphosphate Protein kinase A Protein kinase C Phospholipase C-β Perilipins Proopiomelanocortin 90-kDa ribosomal S6 kinase Receptor tyrosine kinase Src-homologous and collagen protein Sympathetic nervous system Son of sevenless Triglycerides White adipose tissue β-adrenergic receptor Alpha-melanocyte-stimulating hormone.

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(23) INDEX I INTRODUCTION 1. MELANOCORTIN SYSTEM 1.1 1.2 1.3 1.4. MELANOCORTIN PEPTIDES MELANOCORTIN RECEPTORS NATURALLY OCCURRING ANTAGONISTS MELANOCORTIN RECEPTORS ACCESSORY PROTEINS. 2. MCRS INTRACELLULAR TRAFFICKING AND SIGNALING 2.1 CELL SURFACE TARGETING 2.2 CELL SIGNALING. 2.2.1 G PROTEIN-DEPENDENT SIGNALING 2.2.2 G PROTEIN-INDEPENDENT SIGNALING 2.2.3 INTEGRATED MECHANISMS FOR ERK1/2 SIGNALING. 2.3 INTERNALIZATION AND RECYCLING. 2.3.1 INTERNALIZATION AND SIGNALING: SPATIO-TEMPORAL REGULATION OF ERK1/2 PATHWAY. 3. MELANOCORTIN SYSTEM REGULATION OF ENERGY HOMEOSTASIS 3.1 CENTRAL CONTROL OF FOOD INTAKE 3.2 THE ROLE OF MELANOCORTINS ON ADIPOCYTE BIOLOGY. 4. OBJECTIVES AND STUDY OUTLINE 5. REFERENCES. 21 24 24 26 30 31. 32 32 34 34 36 38. 41 42. 44 44 47. 50 53. II PUBLICATIONS. 65. PUBLICAÇÃO I PUBLICAÇÃO II PUBLICAÇÃO III. 67 77 91. III DISCUSSION. UNRAVELING MC5R SIGNALING MC5R AS A NEW PLAYER ON ADIPOCYTE METABOLISM MELANOCORTINS ROLE ON ADIPOCYTE LIPID METABOLISM UNDER OBESITY MELANOCORTIN SYSTEM AS A TARGET FOR ANTI-OBESITY DRUGS REFERENCES. 103 105 108 109 112 115.

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(25) I. INTRODUCTION.

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(27) The modern world is facing a global epidemic of obesity, giving rise to alarming. prevalence rates in many developing and developed countries. In 2008, it was estimated that approximately 1.4 billion adults worldwide were overweight [Body Mass Index. (BMI) ≥25 kg/m2] and 500 million of these were obese [BMI ≥30 kg/m2] (Finucane et al,. 2011). This "globesity” stands as one of the most serious health challenges of the early 21st century all over the World. In Portugal, in 2005, more than half of the population (53.6%) was suffering from excess of body weight and obesity prevalence reached 14.2% (do Carmo et al, 2008). It is estimated that this incidence increased since then and will. continue to in the coming years. Also of much concern is the childhood overweight/obesity incidence in Portugal that reached 30% in 2010, one of the highest and worrisome rates among European countries (Loureiro & Freudenberg, 2012; Yngve et al, 2008).. Overweight and obesity are a consequence of abnormal or excessive fat. accumulation that usually results from an imbalance between food intake and energy. expenditure. Whether obesity integrates or not the concept of disease was a controversy. that lasted for several years (Heshka & Allison, 2001). However, obesity is now. recognized as a chronic and systemic inflammatory disorder and is definitely a major risk factor for many diseases such as heart disease, stroke and other cardiovascular conditions, type 2 diabetes, osteoarthritis and some types of cancers (Pi-Sunyer, 2002).. Therefore, this morbid and potentially fatal entity ought to be taken seriously and efficient strategies to control obesity should be considered.. Besides the easy access to energy-dense food with high palatability and the. adoption of sedentary lifestyles that strongly contribute to sprawling the obesity phenotype, its prevalence also depends on biological and physiological factors. Indeed, diets and exercise are extremely important for the treatment of obesity but in some cases, particularly in severely obese patients, lifestyle modification is still ineffective to. variability among individuals also accounts for the success or failure of such strategies.. In fact, eating behavior and body weight are strongly determined by genetic factors that. account for 40% to 70% of an individual’s predisposition to obesity (Barsh et al, 2000; Loos, 2009; Ranadive & Vaisse, 2008). The most prevalent mutations related to an obese. INTRODUCTION. maintain long-term weight loss (Bray, 2008; Xiao & Yang, 2012) because the inherited. 23.

(28) phenotype were found in genes of the melanocortin system (Bray, 2008; Cheung & Mao,. 2012; Farooqi & O'Rahilly, 2008; Ramachandrappa & Farooqi, 2011; Ranadive & Vaisse, 2008). It has been known for many decades that the senses of appetite and satiety are under control of the central nervous system (CNS), with the hypothalamic. melanocortinergic system having an important anorexigenic role. Alterations on these mechanisms compromise the control of body weight and lead to obesity. Consequently, the melanocortin system at the CNS became an hallmark in the regulation of food intake. and a focus for the development of several anti-obesity drugs by the pharmaceutical industry. However, such efforts have not so far reached the clinic because of a variety of. side-effects indicating that more direct and specific approaches are needed for the pharmacological treatment of obesity. One of this can be the targeting of peripheral. tissues highly implicated in the development of an obese phenotype, like the adipose. tissue. Although the mechanisms were poorly investigated, melanocortins act directly on adipose tissue in order to promote fat mobilization and weight loss (Blüher et al, 2004;. Pierroz et al, 2002; Strader et al, 2007). A detailed study of the molecular and cellular. mechanisms activated by melanocortins will provide new perspectives for the modulation of adipocyte physiology, a promising issue for future designing of new drugs to combat obesity.. 1. MELANOCORTIN SYSTEM The melanocortin system is composed of: (1) the melanocortin peptides, which. include the adrenocorticotropic hormone (ACTH) and alpha, beta and gamma-. melanocyte-stimulating hormones (α, β, γ-MSH); (2) five different cell membrane. receptors named melanocortin receptors (MCRs); (3) two naturally occurring antagonists, the agouti and the agouti-related protein (AgRP) and also (4) the melanocortin 2 receptor accessory proteins (MRAP and MRAP2). 1.1 MELANOCORTIN PEPTIDES All melanocortins derive from post-translational processing of the common. polypeptide precursor proopiomelanocortin (POMC) (Figure 1). POMC gene sequence. was firstly described in bovine pituitary in 1979 (Nakanishi et al, 1979) and shortly thereafter this gene was cloned from a variety of other animals, including humans. (Chang et al, 1980; Takahashi et al, 2001; Takeuchi et al, 1999). Although it was initially. 24. assumed that POMC and POMC-derived peptides were originated from pituitary only,.

(29) high expression levels were later noticed in the arcuate nucleus (ARC) of the. hypothalamus (Bicknell, 2008; Cowley et al, 2001). Additionally, they were found at lower levels in other regions of the brain and in a wide range of peripheral tissues. including skin, placenta, testis, thyroid, adrenal gland, kidney, pancreas, liver, heart,. gastrointestinal tract, lung and immune system (Bicknell, 2008; DeBold et al, 1988a; DeBold et al, 1988b; Millington et al, 1999; Slominski et al, 2000; Smith & Funder, 1988; van der Kraan et al, 1999).. The expression of POMC in different tissues among the entire body surely. accounts for the diversity and wide array of physiological roles that has been frequently. attributed to the melanocortin system. Originally known for the ability to stimulate melanocyte pigmentation and adrenal steroidogenesis, melanocortins are also involved in anti-inflammatory and antipyretic actions; pain modulation and neuronal. regeneration; improvements on learning, attention and memory; regulation of exocrine. gland secretion; modulation of sexual behavior; control of cardiovascular function; mediation of adipocyte physiology and regulation of food intake and energy expenditure. (Bertolini et al, 2009; Catania et al, 2004; Catania et al, 2010; De Jonghe et al, 2011; Garfield et al, 2009; Getting, 2006; Greenfield, 2011; Mountjoy, 2010a; Mountjoy & Wong,. 1997; Pandit et al, 2011; Wikberg et al, 2000).. The POMC gene comprises 3 exons but only exons 2 and 3 are translated,. encoding a 31-36 kDa highly conserved protein with similar structural organization and. tissue-expression pattern across vertebrate species (Bicknell, 2008; Catania et al, 2004; Dores & Baron, 2011; Pritchard et al, 2002). Biologically active melanocortins are produced by successive proteolytic cleavage of POMC, in a tissue-specific manner, by a. family of prohormone-convertases (PCs), of which, PC1 (also known as PC3) and PC2 are of particular importance (Benjannet et al, 1991; Seidah et al, 1999; Thomas et al, 1991). (Figure 1). The tissue pattern expression of PC1 and PC2 may specifically define which melanocortin is produced in each tissue. PC1 cleaves POMC generating ACTH, whereas. PC2 is required for the production of the other melanocortins α, β and γ-MSH (Benjannet et al, 1991). The anterior pituitary seems to have no inherent PC2 activity (Seidah et al,. 1999) thus yielding ACTH as the principal POMC-derived peptide. In contrast, the expression of both PC1 and PC2 within the intermediate lobe of pituitary, hypothalamus. and peripheral tissues leads to the production of all melanocortins (Bicknell, 2008; found in blood circulation are mainly from pituitary origin, but in adult humans, the. intermediate lobe of pituitary is vestigial which implicates that α, β and γ-MSH should be. mainly produced by non-pituitary human tissues (Bicknell, 2008; Gibson et al, 1994).. However, physiologically significant expression levels of POMC were observed only in a. INTRODUCTION. Catania et al, 2004; Pritchard & White, 2007). It is generally assumed that POMC peptides. 25.

(30) limited number of peripheral tissues like skin, lymphocytes and placenta, where it. appears to locally regulate pigmentation, inflammation and fetal development,. respectively (Bicknell, 2008; Catania et al, 2004; Slominski et al, 2000). In other peripheral tissues, the significance of POMC expression remains unknown. Overall, it is. clear that POMC synthesis, post-transcritional processing and secretion are key determinants for regulating melanocortin signaling. However the extraordinary complexity of these mechanisms underscores the difficulty in determining the precise molecular basis behind its biological effects.. Figure 1. Post-translational processing of proopiomelanocortin (POMC). All melanocortin peptides (red rectangles) derive from successive proteolytic cleavages of POMC polypeptide. The initial steps of POMC processing are mediated by prohormone-convertase (PC) 1 (also named PC3) and generate adrenocorticotropic (ACTH) and β-lipotrophin (LPH). Further processing by PC2 originates β and γmelanocyte stimulating hormone (MSH), corticotropin-like-intermediate lobe peptide (CLIP), β-endorphin (βED) and γ-LPH. Carboxypeptidase E (CPE) activity gives rise to mature α-MSH.. 1.2 MELANOCORTIN RECEPTORS In both CNS and peripheral target cells, melanocortin effects are mediated by the. activation of a 5-member family of melanocortin receptors made up of MC1R, MC2R, MC3R, MC4R and MC5R, named by the order of their cloning. They were all identified. between 1992 and 1994, as a product of separated genes (Chhajlani & Wikberg, 1992; Gantz et al, 1993a; Gantz et al, 1993b; Gantz et al, 1994; Griffon et al, 1994; Labbé et al, 1994; Mountjoy et al, 1992) and, since then, remarkable progress has been made in the. melanocortin research. Collectively they belong to the superfamily of the G proteincoupled receptors (GPCRs), cell membrane proteins with seven transmembrane. domains. MCRs are the smallest known GPCRs, with short amino and carboxyl-terminals,. 26. and a very small second extracellular loop (Catania et al, 2004)..

(31) GPCRs constitute a large group of cell-surface receptors and are one of the most. studied targets of the pharmaceutical industry, accounting for up to 50% of clinical drugs. currently available (Drews, 2006; Hopkins & Groom, 2002; Overington et al, 2006; Salon. et al, 2011). GPCRs were only discovered in the 80s with the cloning of rhodopsin (Kühn,. 1980; Nathans & Hogness, 1983; Nathans & Hogness, 1984) and β 2 -adrenergic (Dixon et. al, 1986) receptors. From then on, more than 800 different GPCRs have been identified. across human genome, mediating the role of a wide variety of hormones, neuropeptides. and other chemicals, and regulating sensory outputs like smell, light and taste (Gether,. 2000). They are classified into 6 classes based on their homology and native ligands but, of these, only 4 are present in multicellular animals: the rhodopsin/β 2 -adrenergic-like. receptors (class A), the adhesion and secretin-like receptors (class B), the metabotropic. glutamate/pheromone receptors (class C) and the frizzled/taste2 receptors (class F). (Gether, 2000; Latek et al, 2012; Schiöth & Fredriksson, 2005). The class A is the largest. one and includes all the MCRs.. Human MCRs exhibit sequence homologies ranging from 67%, between the. MC4R and MC5R, to 42% between MC1R and MC2R (MacNeil et al, 2002). They differ from each other on their binding affinity to the melanocortins and on their tissue distribution (Table 1).. MCR. MC1R. MC2R MC3R. MC4R. MC5R. Agonist affinity. Tissue expression. α-MSH> β-MSH> γ-MSH> ACTH. Melanocytes; keratinocytes; fibroblasts; endothelial cells; macrophages; monocytes; neutrophils; mast cells; B lymphocytes; dendritic cells; astrocytes; microglia; corpus luteum; placenta; testis; pituitary; adipocytes. Pigmentation; Anti-inflammatory. Brain; placenta; stomach; pancreas; duodenum; heart; testis; mammary gland; muscle cells; kidney; macrophages; monocytes; B lymphocytes. Energy homeostasis; Anti-inflammatory Cardiovascular control. ACTH. γ-MSH= α-MSH= ACTH> β-MSH α-MSH≥ ACTH> β-MSH> γ-MSH. α-MSH≥ ACTH> β-MSH>γ-MSH. Function. Adrenal gland; skin; adipocytes. Adrenal steroidogenesis. Brain; sympathetic nervous system; diaphragm; extensor muscles; abdominal wall muscles; adipocytes. Control of food intake and energy homeostasis; Erectile function and sexual behavior; Pain modulation. Exocrine glands (lacrimal, prostate, seminal, pancreatic, preputial, harderian and mammary); adrenal gland; pituitary; kidney; liver; lung; lymph nodes; bone marrow; thymus; testis; ovary; uterus; esophagus; stomach; duodenum; skin; skeletal muscle cells; adipocytes; B lymphocytes; brain. Regulation of exocrine glands secretion; Aggressive behavior; Fatty acid β-oxidation; Immunomodulatory functions. INTRODUCTION. Table 1. Melanocortin receptor family: affinity, distribution and functions. 27.

(32) MC1R is named the “α-MSH receptor” because it was firstly described in. melanocytes (Mountjoy et al, 1992) and melanoma tissue (Chhajlani & Wikberg, 1992). and immediately linked to the α-MSH effect on skin pigmentation. The mammalian skin darkness and hair colour are regulated by melanocortin (especially α-MSH) binding to. MC1R which controls the ratio of eumelanin (black/brown) to pheomelanin (red/ yellow) (Cone et al, 1996). Mutations on human MC1R are associated with red or blond. hair and pale skin (Ducrest et al, 2008; Millington, 2006; Wikberg et al, 2000) suggesting. that different skin types and hair colorations are related to fluctuations on MC1R signaling. MC1R was latter reported in keratinocytes, fibroblasts, endothelial cells,. macrophages, monocytes, neutrophils, mast cells, B lymphocytes, dendritic cells, astrocytes, microglia, corpus luteum, placenta, testis, pituitary and adipose tissue. (Catania et al, 2004; Getting, 2006; Hoch et al, 2007; Starowicz & Przewłocka, 2003;. Wikberg et al, 2000). MC1R expression in several inflammation-related cells has been extensively investigated and related to the regulation of melanocortin-mediated anti-. inflammatory pathways (Catania et al, 2004; Catania et al, 2010). Despite its higher. affinity to α-MSH, MC1R also binds all the other melanocortins (Table 1).. MC2R is the classical “ACTH receptor”. It is unique among MCRs because it has. specificity for ACTH binding, not being activated by any other melanocortin (Schiöth et al, 1996; Veo et al, 2011), and is the only MCR that requires specific accessory proteins to function efficiently (Noon et al, 2002; Roy et al, 2007). MC2R was initially cloned from. human adrenal glands (Mountjoy et al, 1992). It is most abundantly expressed in the. adrenal cortex, where it mediates stress responses by regulating steroidogenesis and consequently glucocorticoid synthesis and release. Mutations in the MC2R gene accounts. for 25% of all cases of familial glucocorticoid deficiency (FGD), a rare autossomal. recessive disorder characterized by severe glucocorticoid deficiency, associated with failure of adrenal responsiveness to ACTH, that cause hyperpigmentation, recurrent. infections and hypoglycemia (Chung et al, 2008; Clark & Weber, 1998; Thistlethwaite et al, 1975). MC2R knockout mice resemble FGD patients and revealed that MC2R is. important for postnatal adrenal development (Chida et al, 2007), in accordance with the potent mitogenic activity of ACTH in adrenal cortex (Ferreira et al, 2007; Hornsby & Gill, 1977). MC2R is also found in the skin (Slominski et al, 1996) and in mouse adipocytes. (Boston & Cone, 1996; Cho et al, 2005; Møller et al, 2011; Norman et al, 2003), where it was suggested to promote lipolysis (Boston, 1999).. MC3R is the only MCR that binds all melanocortins with similar affinity (Table 1).. 28. It was firstly cloned from human genome (Gantz et al, 1993a) and described to be.

(33) predominantly expressed in brain areas (Roselli-Rehfuss et al, 1993). MC3R is also present in placenta, stomach, pancreas, duodenum, heart, testis, mammary gland,. muscle, kidney (Chhajlani, 1996; Gantz et al, 1993a; Wikberg et al, 2000) and in immune. cells (macrophages, monocytes and B lymphocytes) where it has a protective role against. inflammation (Catania et al, 2010; Getting, 2006). MC3R function at CNS is not clear but it seems to be related with the regulation of energy metabolism. Recent data suggest that. MC3R modulates adaptations to restricted feeding and the expression of behaviors that. anticipate nutrient availability (food anticipatory activity) (Begriche et al, 2012; Sutton et al, 2010). MC3R knockout mice are slightly obese with increased fat mass, reduced lean mass and higher ratio of weight gain to food intake (Butler & Cone, 2002; Chen et al,. 2000; Cone, 2006). Human mutations on MC3R gene are frequently related to obesity and type 2 diabetes (Mountjoy, 2010a) and some reports indicate that MC3R. polymorphisms are associated with increased risk of childhood obesity (Feng et al, 2005; Mencarelli et al, 2011; Savastano et al, 2009; Zegers et al, 2011).. MC4R was the second melanocortin receptor to be identified in the CNS (Gantz et. al, 1993b), where it presents a broader expression than MC3R (Mountjoy et al, 1994).. Although MC4R was not found at the periphery by Chhajlani (Chhajlani, 1996), Mountjoy and Wong showed its expression in the sympathetic nervous system (SNS), diaphragm, extensor muscles and abdominal wall muscles (Mountjoy & Wong, 1997). The presence. of MC4R in adipocytes did not reach consensus so far (Chagnon et al, 1997; Hoch et al,. 2007; Smith et al, 2003), but recent data revealed that MC4R is expressed in SNS that. innervates adipose tissue in rodents (Song et al, 2005; Song et al, 2008). Alterations on MC4R signaling are commonly associated with eating disorders ranging from obesity to. cachexia syndromes (Coll 2007, Cone 2007, Butler 2006, Lee 2007, Ellacott 2006). MC4R effects on neuronal control of food intake, body weight and energy balance have been intensively reviewed (Lee 2009, De Jongue 2011, Macneil 2002, Adan 2006, Lee 2007,. Montjoy 2010, Millington 2007, Lam 2007). MC4R knockout mice are hyperphagic with. severe obesity, hyperinsulinemia and hyperleptinemia (Huszar 1997, Butler 2002) and. MC4R mutations are frequently found among human obese patients (Beckers et al, 2010; Lee & Wardlaw, 2007; MacKenzie, 2006; Mountjoy, 2010b). In fact, the most prevalent. monogenic obesity syndrome in human results from MC4R mutations, responsible for up behavior and erectile dysfunction, possibly through sympathetic innervations of the. penis (Martin & MacIntyre, 2004; Wikberg & Mutulis, 2008). Additionally, melanocortin effects on pain modulation are attributed to central MC4R expression (Starowicz et al, 2009; Starowicz & Przewłocka, 2003).. INTRODUCTION. to 6% of morbidly obese individuals (Loos, 2011). MC4R is also involved in sexual. 29.

(34) Finally, MC5R, cloned by several independent groups in 1994 (Gantz et al, 1994;. Griffon et al, 1994; Labbé et al, 1994), is the most ubiquitous receptor among the. melanocortin family with a wide peripheral distribution and higher homology between species. It is expressed in adrenal gland, kidney, liver, lung, lymph nodes, bone marrow,. thymus, pituitary, mammary gland, testis, ovary, uterus, esophagus, stomach, duodenum, skin, skeletal muscle, adipocytes and exocrine glands, including the lacrimal, prostate,. seminal, pancreatic, preputial and harderian (Catania et al, 2004; Chen et al, 1997;. Chhajlani, 1996; van der Kraan et al, 1998; Wikberg et al, 2000). It is also present in the brain, although at low levels (Cerdá-Reverter et al, 2003; Gantz et al, 1994; Griffon et al,. 1994; Labbé et al, 1994). MC5R has higher affinity for α-MSH but also binds the other. melanocortin peptides (Table 1). The analysis of MC5R knockout mice linked this. receptor to the control of exocrine glands secretion. These animals have defects in water. repulsion and thermoregulation that are caused by a decrease in lipid production by. sebaceous glands (Chen et al, 1997). This phenotype was found after a swimming test: MC5R knockout mice remained wet for a longer period of time after being removed from. water as a result of a deficit in sterol ester-type lipids in the coat, which also lead to a. longer lasting hypothermia (Chen et al, 1997). MC5R was also shown to be required for stress-regulated synthesis of porphyrins by the harderian gland and for melanocortin-. regulated protein secretion by the lacrimal gland (Chen et al, 1997). The regulation of stress-induced pheromones secretion by preputial gland during aggressive behavior is. also attributed to MC5R (Caldwell & Lepri, 2002; Morgan & Cone, 2006; Morgan et al,. 2004a; Morgan et al, 2004b). In the adrenal glomerulosa cells, α-MSH is known to. regulate aldosterone synthesis, probably through MC5R (Liakos et al, 1998; Liakos et al,. 2000; Vinson et al, 1983). Besides, MC5R has been related with activation of regulatory. T-lymphocytes during ocular immunity (Lee & Taylor, 2011; Taylor & Namba, 2001; Taylor et al, 2006; Taylor & Lee, 2010), with immunomodulatory functions in B-. lymphocytes (Buggy, 1998) and with stimulation of cytokine secretion in adipocytes (Jun. et al, 2010). In skeletal muscle cells, MC5R was specifically implicated on the regulation of fatty acid oxidation (An et al, 2007), while in adipocytes it has been suggested as a possible mediator of lipid metabolism (Boston & Cone, 1996; Møller et al, 2011).. 1.3 NATURALLY OCCURRING ANTAGONISTS MCRs are distinguishable from all the other GPCRs because they are the only ones. with known endogenous antagonists, the agouti and the AgRP, emphasizing the complex. 30. network around MCRs regulation..

(35) The agouti protein was known to control skin pigmentation several decades. before it was cloned by the discovery of the lethal yellow (Ay) mice with a natural. mutation in the agouti locus leading to ectopic expression of agouti in almost all tissues, which is characterized by yellow coat colour, hyperphagia, hyperinsulinemia and obesity. (for review see Moussa & Claycombe, 1999; Yen et al, 1994). In rodents, agouti is. expressed only in skin, whereas its homologue in humans, named agouti signaling protein (ASP), has a much wider distribution through testis, ovary, heart, foreskin,. kidney, liver and adipose tissue (Smith et al, 2003; Wilson et al, 1995). The pigmentation and obesity phenotypes were made clear when agouti was found to antagonize MC1R and MC4R (Lu et al, 1994). Agouti acts as a competitive antagonist with high affinity to MC1R and MC4R and lower affinity to MC3R and MC5R (Dinulescu & Cone, 2000).. The AgRP was cloned based on the similar homology to agouti and was described. as having higher blocking ability for MC3R and MC4R, little for MC5R and no effect on. MC1R and MC2R (Ollmann et al, 1997; Yang et al, 1999). It is mainly expressed in the hypothalamus and adrenal gland (Ollmann et al, 1997) and at lower levels in testis, lung, kidney (Shutter et al, 1997) and blood (Li et al, 2000). As a potent antagonist of central. melanocortin receptors, it is strongly associated to the control of food intake and energy expenditure (Coll, 2007; Lee & Wardlaw, 2007). Periods of fasting promote the release of. AgRP from hypothalamic neurons and its binding to MC4R to inhibit satiety signals. (Ebihara et al, 1999; Stanley et al, 2005; Wynne et al, 2005). Transgenic mice with ubiquitous overexpression of AgRP present obese phenotype similar to Ay mice, but. without alterations in pigmentation (Ollmann et al, 1997). Ablation of AgRP neurons in leptin-deficient obese mice decreases food intake and restores the normal body weight (Wu et al, 2012). In humans there is no evidence for abnormal elevation of AgRP activity,. but there are some reports of SNPs (single nucleotide polymorphisms) in AgRP gene associated with reduced fat mass (Marks et al, 2004; Vink et al, 2001).. 1.4 MELANOCORTIN RECEPTORS ACCESSORY PROTEINS Similarly to other GPCRs, melanocortin receptors also interact with accessory. proteins in order to properly reach the cell surface. The presence of interacting factors. trafficking of MC2R to cell surface when the receptor was expressed in cells that lack endogenous expression of the melanocortin system interveners (Noon et al, 2002).. MRAP, a transmembrane protein, was first identified in 3T3-L1 adipocytes and initially. named fat tissue low-molecular weight protein (Xu et al, 2002). Afterward, Metherell et. INTRODUCTION. for MCRs function was first suggested by Noon et al, who failed to obtain a correct. 31.

(36) al identified this protein as a potential candidate for causing FGD in patients with normal. MC2R and, for this reason, it was renamed as melanocortin 2 receptor accessory protein (MRAP) (Metherell et al, 2005).. Two isoforms of human MRAP (MRAPα and MRAPβ) are produced by alternative. splicing. They share the same N terminus and transmembrane domain but are highly. divergent on their C terminus (Metherell et al, 2005). MRAP was further established as. essential for MC2R trafficking and signaling (Chung et al, 2008; Cooray et al, 2008; Roy et al, 2007; Sebag & Hinkle, 2009a; Sebag & Hinkle, 2009b), a reason why 20% of FGD type. 2 patients have mutations in MRAP gene (Cooray & Clark, 2011). Later on, a closely-. related protein, named MRAP2, was found in adrenal gland and hypothalamus and seemed to function similarly to MRAP (Chan et al, 2009; Sebag & Hinkle, 2009b). MRAPs. have a dual effect on MCRs trafficking and signaling: MC2R requires MRAPs expression (Cooray et al, 2008; Roy et al, 2007; Sebag & Hinkle, 2007; Webb et al, 2009) whereas all the other MCRs traffic efficiently to plasma membrane in the absence of MRAPs.. However, when MRAP is present it impairs MC4R and MC5R trafficking to plasma. membrane while having no effect on cell surface expression of MC1R and MC3R (Chan et al, 2009; Sebag & Hinkle, 2009a). Intriguingly, MRAPs act as negative regulators of MC1R, MC3R, MC4R and MC5R signaling (Chan et al, 2009).. 2. MCRS INTRACELLULAR TRAFFICKING AND SIGNALING 2.1 CELL SURFACE TARGETING Correct translation, maturation and assembly through secretion pathway. represent the first step in intracellular trafficking of GPCRs and determine its proper cell surface expression and function at plasma membrane.. The lifecycle of all GPCRs, as many other membrane proteins, starts with their. synthesis in endoplasmic reticulum (ER). Once correctly folded, the receptors are able to move with ER-derived COPII transport vesicles to the Golgi for further maturation and. subsequent export to cell surface (Dong et al, 2007). During secretion pathway, several. pos-translational modifications (e.g. glycosylation, ubiquitination and oligomerization). take place before the receptors targeting to plasma membrane. Correct folding and assembling are coordinated by GPCR interaction with several regulatory proteins that. 32. ensure a high quality control of newly synthesized receptors. These proteins also.

(37) recognize misfolded receptors, which are no longer able to address the cell membrane. and are targeted for proteasome degradation (Jean-Alphonse & Hanyaloglu, 2011). ER-. resident chaperones, such as calnexin and calreticulin, the 70 KDa heat-shock protein. (Hsp70) family, the receptor activity modifying proteins (RAMPs) and the GTPases family of Rab and Sar1/ARF function broadly to facilitate folding and ER export of. numerous GPCRs (Dong et al, 2007; Wang & Wu, 2012). It was recently shown that. Hsc70, the cognate cytosolic Hsp70 protein, promotes cell surface expression of intracellular retained MC4R mutants (Meimaridou et al, 2011). The authors demonstrate that the obesity-related MC4R mutants S58C, P78L and D90N have reduced traffic to plasma membrane caused by protein misfolding and retention at the ER. In cells. expressing these MC4R mutants, Hsc70 overexpression restores MC4R export from ER to cell surface and promotes its signaling (Meimaridou et al, 2011).. Additionally, other chaperones were found to act selectively in some types of. GPCRs to regulate trafficking and cell surface expression (Dong et al, 2007; JeanAlphonse & Hanyaloglu, 2011). As mentioned previously (section 1.4), MRAP proteins interact specifically with all five MCRs, but are only essential for MC2R targeting to cell. surface (Chan et al, 2009; Webb & Clark, 2010). MRAPs assist MC2R targeting to cell membrane by facilitating ER export and further post-translational processing at Golgi. apparatus (most probably glycosylation) (Sebag & Hinkle, 2007). Besides MRAPs, other proteins were identified as potential accessory proteins for MCRs, namely attractin,. attractin-like protein (ALP) and mahogunin ring finger 1 (MGRN1) (Cooray & Clark,. 2011; Cooray et al, 2011; He et al, 2003). MGRN1 contains a “really interesting new gene”. (RING) finger domain characteristic of the E3 ubiquitin ligases. In fact, ubiquitination is a frequent modification of GPCRs important for their targeting for proteasomal degradation, but also regulates GPCRs signaling, internalization and lysosomal degradation (Dores & Trejo, 2012; Marchese & Trejo, 2013; Shenoy, 2007). It was. recently demonstrated that MGRN1 impairs MC1R and MC4R function by competition. with the Gs subunits inhibiting receptor functional coupling to the cyclic adenosine. monophosphate (cAMP) pathway (Pérez-Oliva et al, 2009). These authors also suggested. that signaling inhibition by MGRN1 occurs independently of receptor ubiquitination or. internalization and might be specific for the MCR subfamily of GPCRs since it is not observed for the β 2 -adrenergic receptor (Pérez-Oliva et al, 2009). However, the role of. degradation rather than with signaling since MC2R became ubiquitinated in the presence of MGRN1 but did not exhibit differences in cAMP production (Cooray et al, 2011).. INTRODUCTION. MGRN1 on MC2R function was postulated to be related with trafficking and/or. 33.

(38) Similar to other GPCRs, all melanocortin receptors oligomerize although direct. evidence is lacking on whether this oligomerization is required to cell surface targeting. In general, dimerization of GPCRs in the ER is an early event of receptor biosynthesis. (Drake et al, 2006; Dupré et al, 2006). Accordingly, in the absence of MRAP, MC2R is able to homodimerize (Sebag & Hinkle, 2009a) but is retained in the ER (Sebag & Hinkle, 2007; Sebag & Hinkle, 2009b). Conversely, MRAP inhibits formation of MC5R dimers and. disrupts its trafficking to plasma membrane suggesting that MC5R monomers may be trapped intracellularly (Sebag & Hinkle, 2009a). Moreover, MC1R undergoes constitutive. homodimerization before reaching the plasma membrane, most likely in the ER (Mandrika et al, 2005; Sánchez-Laorden et al, 2007; Sánchez-Laorden et al, 2006; Zanna. et al, 2008). MC3R form homodimers but can also heterodimerize with MC1R (Mandrika. et al, 2005). More recently, heterodimerization of MC3R with ghrelin receptor (GHSR),. both expressed in the hypothalamic ARC, was found to enhance MC3R signaling efficacy otherwise decreasing GHSR signaling capacity (Rediger et al, 2011), a possible strategy to control body weight. MC4R was also shown to heterodimerize with another GPCR. involved in weight regulation, the G protein-coupled receptor 7 (GPR7) (Rediger et al,. 2009). In addition to the constitutive homodimerization of MC4R observed by different groups (Elsner et al, 2006; Nickolls & Maki, 2006), Biebermann et al showed that MC4R. also dimerize with the D90N mutant, a naturally occurring heterozygous mutation of. MC4R found in obese patients (Biebermann et al, 2003). Co-expression of both wild-type. and mutant receptors results in normal cell-surface expression and binding properties. but loss of signal transduction caused by a dominant-negative effect on wild-type MC4R function (Biebermann et al, 2003). 2.2 CELL SIGNALING Once properly addressed into the plasma membrane, melanocortin receptors are. activated by ligand binding and undergo conformational changes triggering a complex intracellular network to translate extracellular signals into biological responses. In the last decade, remarkable efforts have helped to elucidate some of the molecular mechanisms behind melanocortin signaling, but much remains to be discovered. 2.2.1 G PROTEIN-DEPENDENT SIGNALING. Classically, GPCRs signaling is primary mediated by interaction with guanine. 34. nucleotide-binding regulatory proteins named G proteins. Heterotrimeric G proteins.

(39) consist of an α-subunit that binds GDP and a non-dissociated complex composed by βγsubunits. Receptor activation facilitates the coupling of G protein at the cell membrane. promoting the exchange of bound GDP to GTP on α-subunit. In the active state, G protein. dissociates from the receptor as well as βγ heterodimer dissociates from GTP-bound αsubunit. Both are then able to activate primary effectors, like adenylyl cyclase (AC) or. phospholipase C-β (PLCβ) to further modulate the activity of a variety of second messengers. Signaling is terminated with the hydrolysis of GTP and subsequent re-. association of inactive GDP-bound α-subunit with βγ complex waiting for a new cycle of receptor activation.. According to the sequence homology and functional similarities of G protein α-. subunits, they are divided into four families, Gs, Gi/o, Gq/11 and G12/13 (Wettschureck & Offermanns, 2005). The signaling cascade generated by G protein activation is. particularly dependent from the subtype of G protein that couple to the receptor. For. example, the protein Gs interacts with AC thus mediating the increase in intracellular. cAMP levels and subsequent activation of protein kinase A (PKA), whereas Gi protein. inactivates AC and blocks PKA activation (Taskén & Aandahl, 2004; Wettschureck & Offermanns, 2005). Conversely, downstream effectors for Gq/11 comprise inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG), both originated from the hydrolysis of. phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by PLCβ, and their signaling is conveyed. mainly through the activation of protein kinase C (PKC) and Ca2+ pathways. (Wettschureck & Offermanns, 2005). While G12/13 signaling is thought to be mediated. by Rho-GTPases, it also modulates PKA activity by interacting with its scaffold protein PKA-anchoring protein (AKAP) or PKC by generating DAG (May & Hill, 2008;. Wettschureck & Offermanns, 2005; Woehler & Ponimaskin, 2009). Furthermore, all the G protein subtypes are able to modulate the activation of mitogen activated protein kinase. (MAPK) family via numerous mechanisms (Goldsmith & Dhanasekaran, 2007; Gutkind, 2000; Hawes et al, 1995; Kristiansen, 2004; Luttrell, 2003; May & Hill, 2008).. Notwithstanding, GPCRs frequently couple to more than one G protein subtype, thereby. initiating a complex intracellular signaling network rather than a simple cascade sequence (Gudermann et al, 1996; Wettschureck & Offermanns, 2005; Woehler &. Ponimaskin, 2009). For instance, β 2 -adrenergic receptor is able to couple to both Gs and Gi (Duarte et al, 2012), orexin-2 receptor is functionally coupled to Gs, Gi and Gq (Tang et. G12/13 (Marinissen et al, 2003; McLaughlin et al, 2005). Observations like these suggest. that the quantification of specific early messengers like intracellular cAMP for Gs, or Ca2+. for Gq, is no longer sufficient to measure receptor activity, which requires the evaluation of multiple signaling pathways.. INTRODUCTION. al, 2008) and the thrombin protease-activated receptor 1 couples to Gi/o, Gq/11 and. 35.

(40) In addition to the α-subunits, the βγ dimer also has regulatory functions.. Although initially thought to have no relevance on GPCR signaling, it has been demonstrated. that. βγ-subunits. modulate. several. intracellular. pathways. like. phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2),. PKA and PKC (Goldsmith & Dhanasekaran, 2007; Gutkind, 2000; Luttrell, 2003; Woehler & Ponimaskin, 2009).. All melanocortin receptors are functionally coupled to Gs that stimulates the. cAMP/PKA pathway (Gantz & Fong, 2003). However many recent studies have. highlighted a wide diversity of signaling partners downstream of melanocortin receptors. For instance, MC4R can also couple to Gi/o and Gq, switching from second. messengers such as cAMP and Ca2+ and triggering other downstream pathways than PKA, like PLCβ/PKC, PI3K and ERK1/2 (Büch et al, 2009; Chai et al, 2006; Daniels et al,. 2003; Mountjoy et al, 2001; Newman et al, 2006; Vongs et al, 2004). MC3R can couple to Gi/o (Chai et al, 2007), can increase intracellular Ca2+ levels (Mountjoy et al, 2001;. Wachira et al, 2003) and can interact with PKC, ERK1/2, PI3K and Akt pathways (Chai et. al, 2007; Nyan et al, 2008; Wachira et al, 2003). MC2R is also able to signal through Ca2+,. PKC and ERK1/2 besides Gs/cAMP/PKA pathway (Gallo-Payet et al, 1996; Janes et al,. 2008; Kilianova et al, 2006; Le & Schimmer, 2001; Roy et al, 2011; Winnay & Hammer, 2006). Similarly, MC1R induces Ca2+ levels and ERK1/2 activation but it seems to have no. effect on PKC pathway (Herraiz et al, 2011; Herraiz et al, 2012; Mountjoy et al, 2001; Newton et al, 2005). MC5R activation also promotes Ca2+ increase but the involvement of other G protein than Gs was never demonstrated (Hoogduijn et al, 2002).. 2.2.2 G PROTEIN-INDEPENDENT SIGNALING. In the past few years, many GPCRs were found to signal independently from G. proteins involving a broad set of binding molecules that might influence the activation of several intracellular pathways. These molecular partners include GPCR kinases (GRKs),. β-arrestins, Src-family tyrosine kinases, Janus protein kinase/signal transducers and activators of transcription (JAK/STAT) and PDZ domain-containing proteins (Sun et al, 2007).. Members of the JAK/STAT pathway are well known mediators of receptor. tyrosine kinase (RTK) signaling but they have been recently associated to some GPCRs. like angiotensin receptor AT 1 (Marrero et al, 1995) and chemokine receptors (Ferrand et. 36. al, 2005; Vila-Coro et al, 1999). There is also one study that reports the activation of.

(41) JAK/STAT pathway by MC5R in lymphocytes (Buggy, 1998). The mechanism of GPCR-. mediated JAK/STAT activation is unknown but it seems to involve the activation of Srcfamily kinases and G protein-dependent pathways (Sun et al, 2007). a. It is well established that stimulation of GPCRs also activate signals from RTK by. process. termed. transactivation.. Briefly,. activated. RTK. dimerizes. and. autophosphorylates itself on cytoplasmic tyrosine residues, which creates binding sites for adapter proteins like the Src-homologous and collagen (Shc) protein. Thus, RTKs. function as scaffolds to assemble signaling partners at cell membrane. GPCR-mediated RTK transactivation can occur through several different mechanisms. One of the best. described models involves the GPCR-mediated activation of membrane-associated. metalloproteinases that engage the processing of inactive membrane-tethered growth factors, which are then released outside the cell to act as soluble agonists for binding to. autocrine RTK (Ohtsu et al, 2006; Snider & Meier, 2007). Although this inside-out model of transactivation is well established for the epidermal growth factor receptor (EGFR),. the specific mechanisms by which GPCRs transactivate other RTKs, including the. platelet-derived growth factor (PDGFR), vascular epidermal growth factor (VEGFR) and. insulin-like growth factor (IGFR) receptors, are still poorly characterized (Natarajan & Berk, 2006; Waters et al, 2004). Other mechanisms besides the release of ligands are also. documented for GPCR-mediated RTK transactivation, which can involve Src kinases and. can also occur via α- or βγ-subunits (Goldsmith & Dhanasekaran, 2007; Gutkind, 2000;. Waters et al, 2004). Nevertheless, autophosphorylation on tyrosine residues is required for RTK signaling and modulation of several intracellular pathways such as JAK/STAT, PI3K/Akt and ERK1/2 pathways (New & Wong, 2007; Smith & Luttrell, 2006).. Besides RTK transactivation, Src kinases also assist GPCRs signaling in β-arrestin-. mediated pathways which, generally, relay in PI3K and MAPK activation (New & Wong,. 2007; Sun et al, 2007). Arrestins family are comprised by two visual arrestins (arrestin 1 and arrestin 4) expressed only in retina, and two non-visual arrestins, β-arrestin 1 and β-. arrestin 2 (also named arrestin 2 and arrestin 3), expressed in numerous tissues (Luttrell. & Lefkowitz, 2002). β-arrestins were initially known by their role in GPCR. desensitization and internalization acting as negative regulators of cell signaling, but now are recognized as central scaffold proteins for signal transduction of many GPCRs. β-. arrestins can activate the same downstream molecules than G proteins, but through. huge role as scaffolds for ERK 1/2 signaling. Although β-arrestins may regulate signal. termination by promoting receptor endocytosis, a wide variety of GPCRs continue to signal in endocytic vesicles, a matter that will be discussed further below (section 2.3.1).. INTRODUCTION. different mechanisms. They act as adapters for Src-family tyrosine kinases and have a. 37.

(42) 2.2.3 INTEGRATED MECHANISMS FOR ERK1/2 SIGNALING. Although initially found to mediate the mitogenic activity of growth factors. through RTK activation, ERK1/2 signaling is now recognized to result from a crosstalk between distinct pathways activated by a wide range of GPCRs (Figure 2).. Figure 2. Integrated mechanisms for G-protein-mediated ERK1/2 signaling. All G-proteins subtypes are able to promote ERK1/2 activation by orchestrating multiple intracellular pathways. Gs signaling is mainly conveyed through activation of AC/cAMP/PKA pathway, which inhibits C-Raf, but also leads to the activation of Rap1 that relays in B-Raf stimulation of ERK1/2 signaling. Gi inhibits AC activity blocking its inhibitory effect on C-Raf and concomitantly stimulates ERK1/2 signaling by a Ras-dependent mechanism. G12/13 strongly stimulates JNK activity but can also activate ERK1/2 through Ras or by DAG/PKC pathway. Gq can stimulate ERK1/2 via PLCβ/DAG/PKC as well as PLCβ/IP3/Ca2+ signaling mechanisms, either by direct phosphorylation of c-Raf by PKC or by a Ras-dependent manner, which may involve the recruitment of Src. Besides α-subunit, the complex βγ released from α subunit during GPCR activation is also able to promote ERK1/2 signaling through stimulation of PLCβ or PI3K. PI3K usually relays in Akt phosphorylation, but also leads to Src and/or Ras stimulation of ERK1/2. In addition, G-proteins can also mediate the transactivation of RTK through its βγ-subunits. Activation of PI3K leads to Src-mediated receptor tyrosine kinase phosphorylation and subsequent recruitment of Shc, Grb2 proteins and SOS to stimulate Ras activity and ERK1/2 phosphorylation. RTK transactivation mediated by βγ-subunits may also occur through an inside-out model. This mechanism is well described for the epidermal growth factor (EGF) receptor, in which βγ activates matrix metalloprotease (MMP) proteins that cleave the ectodomains of membrane-bound growth factors (HBEGF) to generate soluble EGF ligands that are released from the cell to activate its RTK. Whatever the mechanism of ERK1/2 activation, these kinases are then able to phosphorylate a wide variety of cytoplasmic and nuclear targets. When traslocate to the nucleus, ERK1/2 initiate gene transcription by phosphorylating several transcription factors like CREB and c-Fos.. 38.

(43) ERK1/2, c-Jun N-terminal kinases (JNK) and p38 pathways constitute the three. main members of the large family of MAPKs. MAPKs are well conserved in all eukaryotes. and are activated in response to many different stimuli. ERK 1 and ERK 2 are codified by separated genes but their high homology make them classically designated as ERK1/2 (also known as p44 and p42 MAPK, respectively). ERK1/2 are able to phosphorylate more than 150 different substrates and modulate a broad array of biological functions. like transcription, proliferation, differentiation, apoptosis, survival, cytokinesis, motility, actin and microtubule network, neurite extensions and cell adhesion, having a crucial role on the immune system and heart development, memory formation and metabolic. homeostasis (Krishna & Narang, 2008; Ramos, 2008). Like all members of MAPKs family,. ERK1/2 pathway consists of three kinases activated sequentially by phosphorylation, named Raf, MEK1/2 and ERK1/2. Briefly, the small GTPase Ras recruit the Raf family of. serine/threonine kinases (C-Raf, A-Raf and B-Raf) to the cell membrane, which can be. activated and then be able to phosphorylate MEK1/2 on two serine residues. MEK1/2. are kinases with a dual specificity that bind to inactive ERK1/2 retaining them in the cytoplasm. Once activated, MEK1/2 phosphorylate ERK1/2 on tyrosine and threonine. residues, allowing ERK1/2 to dissociate from the complex MEK1/2-ERK1/2. Activated ERK1/2 can translocate to the nucleus where they phosphorylate serine/threonine residues of a large number of transcription factors like c-Fos, c-Jun, c-Myc, Elk, AP1 and. cAMP-response element binding protein (CREB) (Ha & Redmond, 2008; Krishna & Narang, 2008). Alternatively ERK1/2 may remain in the cytoplasm to phosphorylate a. distinct set of substrates like GRK2 (Pitcher et al, 1999), β-arrestin1 (Lin et al, 1999), 90kDa ribosomal S6 kinases (RSKs), phospholipase A2, and many cytoskeletal and. membrane proteins (Krishna & Narang, 2008). RSK family members also have the ability. to function in different cellular compartments including cytosol, plasma membrane and nucleus (Anjum & Blenis, 2008; Frödin & Gammeltoft, 1999; Murphy & Blenis, 2006).. Mitogen- and stress-activated protein kinases (MSKs), known as RSK-like proteins. because of their high homology to RSKs are also targets of ERK1/2 but in contrast to. RSKs that are predominantly cytosolic in resting cells, MSKs are constitutively localized on the nucleus (Anjum & Blenis, 2008).. GPCR-mediated ERK1/2 activation can be generated by two main mechanisms, G. proteins can induce ERK1/2 phosphorylation through activation of cAMP/PKA pathway,. whereas the Gq/11 can lead to ERK1/2 signaling by a PKC-dependent mechanism (May. & Hill, 2008) (Figure 3). In addition to G proteins, many GPCRs are also known to induce. ERK signaling by RTK transactivation. Autophosphorylation of RTK stimulates Shc to. INTRODUCTION. protein-dependent and G protein-independent pathways. Upon ligand binding, Gs. 39.

(44) recruit the growth factor receptor-bound protein 2 (Grb2) and the guanine exchange. factor (GEF) son of sevenless (SOS) to the RTK promoting their phosphorylation, which. results in Ras activation and ERK1/2 signaling (Hubbard & Till, 2000; Schlessinger,. 2000). Moreover, βγ-subunits released from Gi/o proteins can also modulate RTK. activation of ERK1/2 pathway through PI3K or PLCβ that promote Src stimulation of Shc. and subsequent Ras activation via the GEF SOS (Goldsmith & Dhanasekaran, 2007; May & Hill, 2008). In some cases, βγ-induced PI3K also signals to ERK1/2 by a Src- and Shc-. independent mechanism, in which Grb2 forms a complex with dynamin II that is able to link SOS to Ras (Goldsmith & Dhanasekaran, 2007) (Figure 2).. Current data regarding the molecular mechanisms that relay in ERK1/2. activation by melanocortin receptors are largely scarce and somewhat controversial. Although conveyed by different mechanisms, MC1R, MC2R, MC3R and MC4R are able to. promote ERK1/2 activation both in native and in overexpressing cell systems (Buscà et. al, 2000; Chai et al, 2007; Chai et al, 2006; Herraiz et al, 2011; Herraiz et al, 2012; Janes et. al, 2008; Le & Schimmer, 2001; Roy et al, 2011; Vongs et al, 2004). In human melanoma. cells, the binding of NDP-α-MSH, a synthetic analog of α-MSH, to MC1R induces ERK1/2. signaling by a mechanism independent from cAMP, PKA, PKC or Ca2+ but involving Src. phosphorylation and transactivation of the RTK c-KIT (Herraiz et al, 2011). In H295R. adrenal cells, ACTH binding to MC2R induces a transient ERK1/2 activation. independently from PKA, PKC and Ca2+ (Janes et al, 2008). In this system, ACTH/MC2Rmediated ERK1/2 signaling does not involve transactivation of the RTK EGFR neither Src. phosphorylation but depends on receptor internalization (Janes et al, 2008). Conversely,. in HEK293/FRT cells overexpressing MC2R, ACTH-induced ERK1/2 activation is PKAdependent but does not require cAMP and PKC (Roy et al, 2011). γ-2-MSH binding to. MC3R overexpressed in HEK293 cells drives ERK1/2 signaling through a PI3K- and Gi/o-. dependent mechanism (Chai et al, 2007). Finally, NDP-α-MSH stimulation of MC4R. endogenously expressed in hypothalamic cell line promotes transient ERK1/2. phosphorylation through Ca2+/PKC pathway but independently from PKA, Gi/o and PI3K. (Chai et al, 2006). However, in CHO cells stably transfected with MC4R, NDP-α-MSH treatment results in PKA-independent but PI3K-dependent phosphorylation of ERK1/2. (Vongs et al, 2004). Cell specificity and differences in the number of receptors expressed in plasma membrane may account for the different mechanisms described so far.. In what concerns MC5R, signaling studies were lacking and direct evidence for. ERK1/2 activation was only reported in the published studies that constitute this thesis.. 40.