Keywords: angiotensin II, G protein, Ca2+signal, tyrosine kinase, receptor phosphorylation, receptor internalisation, candesartan
*Department of Physiology,
Semmelweis University Medical School, Budapest, Hungary
†Department of
Molecular and Biochemical Pharmacology, Institute of Molecular Biology and Biotechnology, Free University of Brussels (VUB), Sint-Genesius Rode, Belgium
§Endocrinology and
Reproduction Research Branch,
National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, USA
Correspondence to: Dr László Hunyady Department of Physiology,
Semmelweis University Medical School, H-1444 Budapest 8, PO Box 259, Hungary
Tel: +36 1 266 2755 (ext 4041) Fax: +36 1 266 6504 E-mail: Hunyady@
Introduction
Angiotensin II (Ang II) is an octapeptide hormone that binds to two distinct heptahelical receptors, the AT1- and AT2-receptors. The AT1-receptor, which has structurally and functionally similar AT1A and AT1Bsubtypes in rodents, mediates the crucial physiological and pathophysiological actions of Ang II on blood pressure regulation, vas-cular tone and salt-water balance.1,2 Early studies
on the therapeutic use of peptide angiotensin receptor antagonists were unsuccessful owing to the partial agonist activity of these compounds.3,4
Since the discovery of non-peptide angiotensin receptor blockers, which selectively recognise the AT1-receptor and have no agonist activity, the AT1 -receptor has become a major therapeutic target in the management of hypertension and other car-diovascular diseases.2,5
Binding of Ang II to the AT1-receptor causes activation of phospholipase C via the Gq/11family of G proteins, initiating inositol phosphate responses, Ca2+ signal generation and protein
kinase C activation.4This second messenger
sys-tem is the main intracellular pathway that medi-ates the physiological effects of Ang II via AT1 -receptor activation.These include smooth muscle contraction, aldosterone secretion, and the control of ion transport in renal tubule cells. More recent studies have revealed that Ang II has important effects on the normal and pathological growth of its target cells, and on the remodelling of cardiac and vascular cells.4,6-8 Some of these effects are
attributable to Ang II-induced calcium signalling,9
but it has become evident that multiple forms of signal transduction can participate in such responses. These pathways include activation of receptor and non-receptor protein tyrosine
kinas-vation is the agonist-induced internalisation of the ligand-receptor complex.11,12Although other
path-ways have also been implicated, internalisation of the AT1-receptor occurs predominantly by endo-cytosis via clathrin-coated pits.12-14 Most of the
available data indicate that internalisation of G protein-coupled receptors (GPCRs) is important for the regulation of receptor sensitivity in at least two ways. First, it reduces the number of available cell surface receptors, and secondly, it facilitates resensitisation of plasma membrane receptors that have been desensitised by GPCR kinase (GRK)-mediated phosphorylation.12,15 It has been
suggested that dephosphorylation of GPCRs (e.g., the β2-adrenergic receptor) occurs within the endosomes after receptor endocytosis, and that the subsequent recycling of the resensitised receptor to the cell surface maintains signal gen-eration.16,17In addition to its role in the regulation
of receptor sensitivity, the internalisation process has been proposed to contribute to the initiation of multiple intracellular signalling pathways. For example, internalisation of the receptor appears to be required for extracellular signal-regulated kinase (ERK) activation by the β2-adrenergic receptor and certain other GPCRs.12,18 However,
ERK activation by many GPCRs is independent of receptor internalisation.12
In addition to advances in understanding the complexity of the AT1-receptor-activated signal transduction pathways, recent studies have begun to identify the molecular mechanisms that occur during receptor activation. Improved understand-ing of the structural requirements for the opera-tion of signal transducopera-tion pathways should pro-vide clues about the sequence of events that occur during Ang II action. The results of these
Structural requirements for signalling and regulation of
AT
1-receptors
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March 2001 Volume 2
Based on these data, Karnik and co-workers have developed a model of AT1-receptor activation dur-ing Ang II binddur-ing.20,21The side-chains of Arg2, His6,
and Pro7do not contribute to the agonist
proper-ties of the ligand, but may be important for the optimal positioning of the critical Tyr4 and Phe8
side-chains. As expected, the major contacts that activate the receptor are formed by Tyr4and Phe8
of the ligand (Figure 1). It has been convincingly demonstrated that the carboxyl group of Phe8
interacts via a salt bridge with Lys199 of the
recep-tor molecule during ligand binding.21,22The
posi-tions of these amino acids are shown in Figure 2. A K199Q mutation of this residue that eliminates its charge caused a marked loss of binding affinity for Ang II, similar to that observed when the nega-tive charge of the carboxyl group of Phe8 of the
hormone is masked by an amide group.22,23
Non-peptide ligands of the AT1-receptor also interact with Lys199. It is interesting to note that insur-mountable AT1-receptor antagonists (e.g., can-desartan) also have a carboxyl group that forms a salt bridge with Lys199.24
The interaction of the carboxyl group of Phe8
with Lys199 is not critical for AT1-receptor activa-tion, but is very important for the optimal posi-tioning of the side-chain of Phe8 because it
sta-bilises the position of this side-chain over the Pro7
-Phe8peptide bond.21The interaction of His256 of
the receptor with the side-chain of Phe8is critical
for receptor activation, and mutations of His256 (H256Q and H256A) cause impaired inositol phos-phate signalling.22Studies using site-directed
muta-genesis of the receptor and analogues of Ang II suggest that a direct interaction between the side-chain of Tyr4and that of Asn111 of the receptor is
also critical for receptor activation.21,25It has been
proposed that the aromatic ring of Tyr4 acts as a
hydrogen bond acceptor from the Asn111 residue of the receptor during this interaction.21,26
During agonist binding, Tyr4and Phe8serve as
‘agonist switches’, and promote relaxation of the receptor’s conformation from its inactive basal state to a partially activated state.21 Mutations of
Asn111 cause constitutive activation of the recep-tor by eliminating the constraining intramolecular interaction of this residue, leading to a similar par-tially activated state of the receptor.21,27,28 The
par-tially activated state induces a conformational change in the Ang II binding pocket, which causes
Figure 1 Critical interactions between Ang II and the AT1-receptor for activation of the signal transduction.The helices are positioned based on the recently elucidated molecular structure of bovine rhodopsin.81Distortion of
helices caused by proline residues are not shown.
Figure 2 Two-dimensional model of the rat AT1A-receptor. The numbering of the amino acids discussed in the present paper is shown.White letters on black background represent conserved residues.
Angiotensin II
AT1
misalignment of the residues required for losartan binding and improves the positioning of residues required for agonist binding. Thus, this state is also characterised by decreased affinity for losartan and increased affinity for agonists. Subsequently, the peptide backbone of Ang II activates the unconstrained receptor to the fully activated state.21,25 Interestingly, studies with [Sar1,Ile4,Ile8]
Ang II have demonstrated that docking of the side-chains of Tyr4and Phe8of the hormone to Asn111
and His256 of the receptor, respectively, is not required for the stimulation of receptor phospho-rylation, despite the crucial importance of these interactions for G protein activation.20,21
The above data indicate that previous models of GPCR activation based on the hypothesis that
tively active phenotype.27 However, other studies
were unable to reproduce this property of the N295S and other Asn295 mutant AT1-receptors, and the role of this residue in this process has been questioned.29-31 Alternatively, a preliminary
model of the AT1A-receptor, and experimental find-ings with mutant AT1A-receptors, suggest that Asn111 forms a hydrogen bond with Tyr292 in the resting state of the receptor. Agonist binding would disrupt this interaction to allow Tyr292 to interact with the conserved Asp74 residue.28,32
The NPX2-3Y motif in the seventh transmem-brane helix is conserved in most GPCRs.33Based
on its similarity to the NPXY internalisation signal of the LDL receptor, an early study on the β2 -adren-ergic receptor suggested that this motif may act as an internalisation signal for GPCRs.34 However,
additional studies have clarified that this sequence is not an internalisation motif in the AT1-receptor and other GPCRs.35-38 It is probable that
substitu-tion of the conserved tyrosine residue in this sequence of the β2-adrenergic receptor impaired its internalisation because it affected phosphory-lation of the receptor.34,35Substitutions of Asn298
and Tyr302 in the NPX2-3Y sequence of the AT1 -angiotensin receptor cause parallel impairment of inositol phosphate signal generation and receptor internalisation.37,38 Based on these findings, it is
likely that the NPX2-3Y sequence of the AT1 -recep-tor has a role in its agonist-induced conformation-al rearrangement during Ang II binding.
Recent studies have suggested that the presence of less conserved polar residues, such as Ser115 in the third transmembrane helix39and Asn294 in the
seventh helix,30is also required for receptor
activa-tion, but the exact roles of these residues have not been identified. Additional studies are required to elucidate the complex rearrangement of the AT1 -receptor structure during -receptor activation, but it is likely that other conserved residues in the trans-membrane helices (e.g., Asn46, Tyr215) also con-tribute significantly to this process.
Multiple receptor states during AT1-receptor
activation
The agonist-induced conformational change of the receptor leads to stimulation of its pleiotropic messenger systems and activates mechanisms that regulate the function of the receptor, such as receptor phosphorylation and internalisation.
Figure 3 Inositol phosphate responses in transiently transfected CHO cells. CHO cells were transfected with a cDNA of the rat AT1A-receptor, and prelabelling of the cells with [3H]inositol was carried out as previously described.30
The cells were pretreated in the presence of 10 mM LiCl for 30 minutes at 37°C, and incubated in the absence (control) or presence of 1 µM Ang II (Ang II), 1 µM [Sar1,Ile8]Ang II (SarIle) or 1 µM candesartan (cand) for
20 minutes at 37°C. Extraction of inositol phosphates and analysis of labelled inositol phosphates was performed as described by Vauquelin et al.82and Hunyady et al.,30
respectively.The data are shown as means±SEM of the inositol phosphate labelling relative to control for three experiments performed in duplicate. Only the response after Ang II treatment was significantly different (p<0.01).
Contr Ang II SarIle Cand
0.5 1.0 1.5 2.0 2.5 3.5
InsP
2
+InsP
3
response
3.0
0
*
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receptor signalling and endocytosis are demon-strated in Figures 3 and 4, respectively. Ang II stim-ulates inositol phosphate formation by the AT1A -receptor transiently expressed in CHO cells (Figure 3). Under the same conditions candesartan, an insurmountable non-peptide antagonist of the AT1 -receptor, has no effect on inositol phosphate accu-mulation, whereas [Sar1,Ile8]Ang II has minimal
ago-nist activity in these cells (Figure 3). Endocytosis of these ligands was studied in CHO cells that were transiently transfected with a fusion protein of rat AT1A-receptors with a highly fluorescent mutant of the green fluorescent protein (EEGFP)43(Figure 4).
Similar to the cells treated with Ang II (5 minutes, 37°C, Figure 4B), endocytosis of the AT1A -recep-tor/EEGFP fusion protein was clearly stimulated in cells treated with [Sar1,Ile8]Ang II (5 minutes, 37°C,
Figure 4D).As demonstrated in Figure 4F, even more prolonged (15 minutes) incubation with candesar-tan did not cause endocytosis of the receptor.These data demonstrate that the agonist activity of [Sar1,Ile8]Ang II is different for signal generation
and receptor internalisation, and are in accordance with the notion that conformational requirements of the receptor for G protein activation and recep-tor internalisation are different.
A very recent study, using constitutively active AT1A-receptor mutants and peptide analogues of Ang II, has provided more specific evidence for the existence of multiple conformational states during AT1-receptor activation, and has demon-strated that different conformations are required
for G protein activation, receptor phosphorylation and internalisation.25 Some of these differences
can obviously be caused by the fact that different regions of the intracellular surface of the receptor interact with the signalling molecules, with pro-teins that mediate receptor internalisation, and with receptor kinases and other regulatory mole-cules. As detailed below, internalisation of the AT1-receptor occurs in the absence of G protein coupling, and some internalisation-deficient AT1 -receptors exhibit normal inositol phosphate signalling after stimulation with Ang II.12,44
Substitutions of residues 234–240 in the C-terminal portion of the third intracellular loop of the AT1A -receptor with the corresponding segments of the
α1B-adrenergic,β2-adrenergic, and the AT2-receptors produce chimeric receptors that differ in their abil-ity to activate inositol phosphate responses, recep-tor internalisation, and mitogenic signalling.45These
data, in accordance with studies on mutant AT1 -receptors detailed below, demonstrate the different structural requirements on the intracellular surface of the activated receptor to initiate G protein acti-vation and other agonist-induced mechanisms.
G protein activation
Members of the Gq/11family of G proteins are the major transducing proteins coupled to the AT1 -receptor, and most studies on the structure-func-tion relastructure-func-tionships of the AT1-receptor have investi-gated their dependent functions. However, the
Figure 4 Endocytosis of an AT1A-receptor-EEGFP fusion protein in transiently transfected CHO cells. CHO cells were plated onto poly-L-lysine-coated glass coverslips at a density of 5x104cells per dish and cultured for three days before the
experiment.The cells were transfected using Fugene 6 (3 µg/ml, 48 hours) with a rat AT1A-receptor cDNA fused to a highly fluorescent mutant of GFP (EEGFP) described earlier by Tarasova et al.43The cDNA of EEGFP was attached in frame to the
C-terminal end of the receptor. The cells are shown before (A, C, E) and after treatment with 1 µM Ang II (B), 1 µM [Sar1,Ile8] Ang II (D), or 1 µM candesartan (F) for 5, 5, or 15 minutes, respectively, at 37°C.The fluorescence of the cells was
AT1-receptor can also activate members of the Gi and G12/13 families in certain tissues.4 The
main intracellular regions of GPCRs that are required for G protein activation are the second and third intracellular loops.4,44-49 Residues in the
membrane-proximal part of the cytoplasmic tail have also been implicated in this process,50 but
other studies have suggested that this region is more relevant to the regulation of expression of the AT1-receptor.51
It is believed that, similar to other GPCRs, the conserved Asp125-Arg126-Tyr127 (DRY) sequence of the AT1-receptor at the N-terminal portion of the second intracellular loop is a major determi-nant of G protein activation.46A recent study has
demonstrated that mutations of amino acids Tyr127 to Ile130 also impair inositol phosphate signal generation.52 Earlier studies proposed that
several polar amino acids in the second intracellu-lar loop of the receptor (Lys135, Ser136, Arg137, Arg139 and Arg140) are also required for G protein activation.46 On the other hand,
substitu-tion of these residues had no major effect on the internalisation kinetics of the receptor.53
In the third intracellular loop, both the N- and C-terminal regions have been implicated in G protein activation.44,45,47,49 At the boundary of
helix V and the third intracellular loop,Tyr215 and Leu222 have been shown to be the major struc-tural requirements of G protein activation and inositol phosphate signal generation.48,54,55
Interestingly, mutations of these residues have a parallel inhibitory effect on the internalisation kinetics and inositol phosphate signal generation of the receptor. Since it is unlikely (although not impossible) that the same residue interacts with the G protein and with proteins required for receptor internalisation, these mutational studies suggest that this region of the receptor may have a role in receptor activation (e.g., by partici-pating in the network of intramolecular interac-tions that stabilise the active conformation of the receptor).
Studies with chimeric AT1A-/AT2-receptors have suggested that the C-terminal portion of the third intracellular loop is also an important determinant of G protein activation by the AT1-receptor.Substitution of charged residues (Lys240, Lys242, Arg244, Asp246 and Asp247) in the C-terminal region of the third intracellular loop of the AT1A-receptor caused
inhibi-Tyrosine kinase activation
Activation of the AT1-receptor by Ang II stimulates the activity of receptor tyrosine kinases (e.g., EGF-receptor, PDGF EGF-receptor, IGF-I receptor and Axl) and non-receptor tyrosine kinases, including c-Src, Ca2+-dependent tyrosine kinases (e.g., PYK2), focal
adhesion kinase (FAK) and Janus kinases (e.g., JAK, TYK). The Ang II-induced activation of tyro-sine kinases leads to downstream signalling mole-cules, including STAT proteins, Ras, ERK, c-fos, Akt/protein kinase B, and p70 S6 kinase. The AT1 -receptor has no intrinsic tyrosine kinase activity, and activation of receptor and non-receptor tyro-sine kinases by the AT1-receptor must occur via protein–protein interaction, or may be mediated by second messengers, such as Ca2+.4,7,8
The AT1-receptor has been shown to activate the JAK/STAT pathway, which was initially found to mediate the growth-promoting actions of cytokines and growth hormone.56,57JAK2 has been
reported to associate with the receptor, and this interaction is mediated by a Tyr-Ile-Pro-Pro (YIPP) sequence in the C-terminal tail of the AT1 -recep-tor.58 Tyrosine phosphorylation of the YIPP
sequence is not required for activation of this process. The YIPP sequence of the AT1-receptor has been suggested to participate in the activation of other signalling molecules, such as phospholi-pase C-γ59and protein tyrosine phosphatase-1D.60
This pathway must act as a parallel signalling route with G protein activation by the receptor, because elimination of this motif by substituting the Tyr319 residue of this sequence with a stop codon did not interfere with inositol phosphate sig-nalling.61An additional similarity between Ang II
and cytokines, such as IL-2 and tumour necrosis factor-α, is the activation of c-Jun N-terminal kinase (JNK). Small G proteins Rac and Cdc42, reactive oxygen species, and p21-activated kinase (PAK) have been suggested to play a role in this process.10,62,63It is interesting to note that the
struc-tural requirements for activation of ERK, a tyrosine kinase-mediated pathway, differ from those required for activation of the JNK pathway. Whereas deletion of Ala221 and Leu222 in the third intracellular loop of the AT1-receptor inter-feres with G protein activation, receptor internali-sation and ERK activation, this mutant receptor retains the ability to activate JNK.63
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March 2001 Volume 2 Supplement 1
tyrosine kinases (such as PYK2/CAKβ) can also acti-vate c-Src,66and this mechanism may contribute to
Ang II-induced c-Src activation. PYK2 is a member of the FAK family. FAK, a 125 kDa protein tyrosine kinase, is a major component of focal adhesions and it acts in partnership with Src family kinases to reg-ulate cytoskeletal changes.8 The structural
require-ments of FAK activation by the AT1-receptor are also unknown.
An additional important mediator of the AT1 -receptor-mediated effects on cellular growth is the transactivation of tyrosine kinase receptors. Three possible mechanisms have been proposed for trans-activation of the EGF-receptor by the AT1-receptor.7 First, Ca2+- and/or protein kinase C-mediated
activa-tion of Ca2+-dependent tyrosine kinases may
acti-vate Src or JAK family kinases to phosphorylate the EGF-receptor. Secondly, reactive oxygen species may activate protein tyrosine kinases that mediate the EGF-receptor phosphorylation. Finally, a recent study has suggested that metalloproteinase-depen-dent cleavage of the EGF-receptor ligand HB-EGF may activate this receptor during stimulation by GPCRs.67It has been proposed that reactive oxygen
species mediate the stimulatory actions of Ang II on JNK, p38 MAP kinase, and Akt/protein kinase B acti-vation, but the molecular mechanism leading to activation of the NAD(P)H oxidase that generates these molecules is not known.10
Low-molecular-weight G proteins
Tyrosine kinase-mediated autophosphorylation of growth factor receptors may initiate the binding of adaptor proteins, such as Grb2 and Shc, that interact with guanine nucleotide exchange factors (GEFs) to stimulate the activities of low-molecular-weight (small) G proteins, including Ras and Rho. Small G proteins of both the Ras and Rho subfam-ilies play critical roles in growth regulation and in control of the actin cytoskeleton.4,68 Recent
stud-ies have suggested that Gα13 and other αsubunits of G proteins can stimulate Rho by activating p115-RhoGEF or other RhoGEFs.69Ang II has been
reported to activate the G12/13 family of G pro-teins,70 and these G proteins may mediate Ang
II-induced activation of Rho kinase.71 However,
recent studies have suggested that activation of small G proteins by GPCRs may involve more direct mechanisms. Many GPCRs cause activation of phospholipase D via the small G proteins, ARF and RhoA. It has been proposed recently that GPCRs containing the conserved Asn-Pro-Xxx-Xxx-Tyr sequence in their seventh transmem-brane helix, including the AT1-receptor, form func-tional complexes with ARF and RhoA.72
Internalisation of the AT1-receptor
Studies with mutant AT1-receptors have suggested that the structural requirements of AT1-receptor internalisation and signal generation are diver-gent. Substitution of a conserved aspartic acid residue (Asp74) by Asn in the second transmem-brane helix disrupts G protein coupling of the AT1A-receptor,44,73 but the internalisation of this mutant receptor is only slightly impaired.40,44,74
Combination of the D74N mutations with a six amino acid deletion of the cytoplasmic tail pro-duced a mutant AT1A-receptor that showed no detectable G protein coupling, but had only mod-erately impaired internalisation kinetics.44
As detailed above, mutations of several apolar residues in the third intracellular loop of the AT1A -receptor molecule cause parallel impairments of receptor internalisation and signal generation.37,48,54
On the other hand, although internalisation of the AT1-receptor is markedly inhibited by deletion of the cytoplasmic tail of the molecule, there is no concomitant loss of Ang II-induced inositol phos-phate or Ca2+ signal generation.53,61,75-77 Detailed
analyses of the amino acids of the cytoplasmic tail have identified two regions that are involved in AT1-receptor internalisation. Optimal internalisa-tion of the AT1-receptor requires the presence of Leu316 and Tyr319 in the more proximal portion of the cytoplasmic tail.78The proximity of Leu316
to another leucine residue has led to the sugges-tion that these amino acids may act as a dileucine internalisation motif and Tyr319 might be part of a tyrosine-containing internalisation motif. However, the exact role of these amino acids in AT1-receptor internalisation has not been eluci-dated.
The most important determinant of AT1 -recep-tor internalisation is a serine/threonine-rich region in the cytoplasmic tail, which includes a Ser335-Thr336-Leu337 triad.61,78 Recent studies on
mutant AT1-receptors with alanine substitutions have revealed that the Ser335-Thr336 sequence is
rapidly phosphorylated after receptor activa-tion.79,80 Substitution of negatively-charged
residues for the putative phosphorylation sites accelerated the internalisation kinetics of the receptor compared to those of the alanine substi-tutions.80These findings suggest that
phosphoryla-tion of the receptor regulates the kinetics of AT1 -receptor internalisation.This conclusion has been questioned based on recent findings with consti-tutively active (N111A and N111G) mutant AT1A -receptors, which showed strongly impaired recep-tor phosphorylation and normal internalisation kinetics.25However, the conformation of
constitu-tively active AT1-Rs differs from that of the Ang II-activated wild-type receptor,25 and a different
internalisation mechanism might be operative in these mutants.
Summary
multiple conformational states that couple differ-entially to specific effectors.The structural require-ments of the receptor for activation of G proteins, receptor and non-receptor tyrosine kinases, recep-tor phosphorylation, and receprecep-tor internalisation, are apparently different. Additional structure-func-tion studies may help to clarify the exact sequence of signalling events after receptor activation, and should also elucidate the specific roles of other mediators, such as reactive oxygen species and small G proteins in AT1-receptor function.
Acknowledgements
The excellent technical assistance of Katinka Süpeki and Judit Bakacsiné Rácz is greatly appre-ciated. The enhanced GFP (EEGFP) cDNA sequence utilised in this study was kindly provid-ed by Dr Stephen A Wank (NIDDK, NIH, Bethesda, Maryland, USA). This work was supported by AstraZeneca Sweden, a Collaborative Research Initiative grant from the Wellcome Trust (051804/Z/97/Z), and by grants from the Hungarian Ministry of Education (FKFP-0318/1999), the Hungarian Science Foundation (OTKA T-032179), and the Hungarian Ministry of Public Health (ETT 31/2000).
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![Figure 3 Inositol phosphate responses in transiently transfected CHO cells. CHO cells were transfected with a cDNA of the rat AT 1A -receptor, and prelabelling of the cells with [ 3 H]inositol was carried out as previously described](https://thumb-eu.123doks.com/thumbv2/123dok_br/18171770.330024/3.892.171.492.339.630/phosphate-responses-transiently-transfected-transfected-prelabelling-previously-described.webp)
