Synthesis of an Agonistic,Difluoro-Azido Photolabel of Angiotensin II and Labeling of the AT1 Receptor: Transmembrane Domains 3, 6, and 7 Form the Ligand-Binding Pocket

p-Azido-phenylalanine has been frequently used for photoaffinity labeling of target proteins such as the angiotensin receptors. However, chemical studies showed that simple aryl nitrenes first react intramolecularly, forming a semistable cyclic keteneimine and then reacting with nucleophile residues in the target structure like those of lysine and arginine. We synthesized 3,5-difluoro-4-azidophenylalanine where the formation of the keteneimine is prevented and where photoincorporation should be due to nonselective nitrene insertion only. This new amino acid was introduced in position 8 of angiotensin II and compared with the corresponding azidophenylalanine peptide using human AT₁ receptor as target. The new photolabel maintained full agonist activity and a similar yield of photolabeling but without the previously observed gradual hydrolysis. Several selective proteolyses of the labeled receptor indicate that the new photolabel forms three simultaneous contact regions on the hAT₁ receptor, suggestive of a nonselective behavior of the photolabel. A major contact was established in the sixth transmembrane domain but also in the third and seventh domain. Our results are in excellent agreement with those recently obtained from methionine proximity assay studies.     

Implication of the First and Third Extracellular Loops of the μ-Opioid Receptor in the Formation of the Ligand Binding Site: A Study Using Chimeric μ-Opioid/Angiotensin Receptors

Abstract: Recent studies on chimeric μ/δ-, μ/κ- and δ/κ-opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric μ receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric μ receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100-fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than μ receptors, was shown to be responsible for this situation. Substitution of the μ receptor second extracellular loop by that of the angiotensin receptor diminished by ∼10-fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the μ receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.     

Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor.

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.     

The vasoactive intestinal peptide (VIP) alpha-Helix up to C terminus interacts with the N-terminal ectodomain of the human VIP/Pituitary adenylate cyclase-activating peptide 1 receptor: photoaffinity, molecular modeling, and dynamics

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors. We characterized the C terminus-binding site of VIP in the N-terminal ectodomain (N-ted) of the human VPAC1 receptor: 1) The probe [(125)I-Bpa(28)]VIP in which the C-terminal residue (Asn(28)) is substituted by a photoreactive p-benzoyl-l-Phe (Bpa) was used to photolabel the receptor. After receptor cleavage and Edman sequencing, it was shown that Asn(28) of VIP is in contact with Lys(127) in the receptor N-ted. Taking into account previous data, it follows that the C-terminal and central parts of VIP from Asn(28) to Phe(6) lie in the N-ted. 2) A three-dimensional model of the N-ted was constructed, the fold being identified as a Sushi domain with two antiparallel beta-sheets and three disulfide bonds. The nuclear magnetic resonance structure of VIP was then docked into this model by taking into account the constraint provided by photoaffinity experiments with [(125)I-Bpa(28)]VIP. It appeared that VIP runs parallel to the beta3-beta4 antiparallel sheets. 3) We performed molecular dynamic simulations over 14 nsec of the complex between VIP and receptor N-ted and the free N-ted. The structural model of the free N-ted is stable, and VIP tends to further stabilize the N-ted structure more especially in the loops connecting the beta-sheets. These structural studies provide a detailed molecular understanding of the VIP-receptor interaction.     

Docking of linear peptide antagonists into the human V(1a) vasopressin receptor. Identification of binding domains by photoaffinity labeling.

A novel photoactivatable linear peptide antagonist selective for the V(1a) vasopressin receptor, [(125)I][Lys(3N(3) Phpa)(8)]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [(125)I]3N(3)Phpa-LVA (Phalipou, S., Cotte, N. , Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V(1a) receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp(112)-Pro(120) could be considered as the smallest covalently labeled fragment with [(125)I][Lys(3N(3)Phpa)(8)]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [(125)I]3N(3)Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V(1a) receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.     

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.     

Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.     

Determining the environment of the ligand binding pocket of the human angiotensin II type I (hAT1) receptor using the methionine proximity assay

The peptide hormone angiotensin II (AngII) binds to the AT0 (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. A series of 44 transmembrane domain III, VI, and VII X --> Met mutants photolabeled either with 125I-[Sar1,p'-benzoyl-L-Phe8]AngII or with 125I-[Sar1,p'-methoxy-p'-benzoyl-L-Phe8]AngII were purified and digested with cyanogen bromide. Several mutants produced digestion patterns different from that observed with wild type human AT1, indicating that they had a new receptor contact with position 8 of AngII. The following residues form this binding pocket: L112M and Y113M in transmembrane domain (TMD) III; F249M, W253M, H256M, and T260M in TMD VI; and F293M, N294M, N295M, C296M, and L297M in TMD VII. Homology modeling and incorporation of these contacts allowed us to develop an evidence-based molecular model of interactions with human AT1 that is very similar to the rhodopsin-retinal interaction.     

Analysis of the adrenal angiotensin II receptor with the photoaffinity labeling method

The angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, AT) receptor of bovine adrenocortex has been investigated with photosensitive analogues of AT. In a first series of experiments, we have shown that isolated cortical cells secrete aldosterone in a permanent and specific manner if they have been photolyzed in the presence of the photolabel [Sar1,(4'-N3)Phe8]AT. This permanent stimulation is in contrast to the smooth muscle assays where under similar conditions a permanent and specific block was always observed. It is assumed that the irreversible occupation of the AT receptor produces this effect. In a second type of experiment, we have shown that the AT binding site on adrenocortical membranes can be specifically and irreversibly occupied under similar conditions and that this occupation can be prevented in a competitive manner by the presence of nonphotosensitive hormone. Using a radioactive label, [Sar1,(3'-125I)Tyr4,(4'-N3)Phe8]AT, we have identified the AT receptor as a 300-kDa protein by means of gel filtration under nonreducing and nondenaturating conditions. Under reducing and denaturing conditions, a subunit of 60 kDs was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The AT receptor is proposed to be a 300-kDa protein with one binding subunit of 60 kDa.     

Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome.

We report the existence of two structurally distinct forms of the angiotensin receptor AT-1 in the mouse. A Balb/c mouse genomic library was screened by homology screening with a polymerase chain reaction (PCR) amplified probe. Restriction mapping and sequencing of the isolated genes revealed the presence of two receptor isoforms, here named the mouse AT-1a and AT-1b receptors, containing 22 different amino acids. Receptor binding studies performed on COS-7 cells transfected with the two receptors revealed that they had similar binding profiles for angiotensin II, angiotensin III and AT-1 or AT-2 specific antagonists. Because many of the structural differences were in the carboxy terminal putative intracellular domain, we speculate that these isoforms may differ in their regulation, signal transduction, or desensitization mechanisms.     

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.     

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.     

Dominance of type 1 angiotensin II receptor in the nonpregnant and pregnant bovine uterus

The present study was undertaken to characterize, determine and localize angiotensin II receptors in the nonpregnant and pregnant bovine uterus. In addition, the concentration of active renin, which is responsible for the generation of angiotensin, was determined. Autoradiography and angiotensin II receptor binding studies showed that all compartments of the bovine uterus contained high concentrations of angiotensin II receptors. In general, the type 1 angiotensin II receptor (AT1) predominated over the AT2 receptor. In the endometrium, the highest density was found in the caruncles and the AT1 receptor was always predominant. The density of angiotensin II receptors in the endometrium increased at the beginning of pregnancy, but decreased and reached values similar to those in nonpregnant animals near term. In the myometrium, the density of angiotensin II receptors was highest at or near the endometrial-myometrial junction. In this area, the predominant type of angiotensin II receptor in the uterus of cyclic cows varied, whereas the AT1 receptor always predominated during pregnancy. Non-AT1 and non-AT2 binding sites were found in the same locations as the angiotensin II receptors, but at lower densities. With the exception of the pregnant endometrium, all compartments contained higher active renin concentrations than found in plasma, indicating local synthesis of renin. This study demonstrates a difference in the expression of types of angiotensin II receptor in the bovine uterus compared with other species. The high densities of angiotensin II receptors localized in several important areas imply that the renin-angiotensin system participates in regulation of growth and tissue function in the bovine uterus.     

Analysis of the mechanisms underlying the vasorelaxant action of angiotensin II in the isolated rat carotid

It has been suggested that low concentrations of angiotensin II cause vasoconstriction whereas high concentrations evoke vasodilation. Thus, this work aimed to functionally characterize the mechanisms underlying the relaxation induced by angiotensin II at high concentrations in isolated rat carotid rings. Experiments using standard muscle bath procedures showed that angiotensin II (0.01-3 μM) concentration dependently induces relaxation of phenylephrine-pre-contracted rings. No differences between intact or denuded endothelium were found. The angiotensin II-induced relaxation was strongly inhibited by saralasin, the non-selective antagonist of angiotensin II receptors but not by the selective antagonists of AT₁ and AT₂ receptors, losartan and PD123319, respectively. However, A-779, a selective angiotensin-(1-7) receptor antagonist, reduced the relaxation induced by angiotensin II. Administration of exogenous angiotensin-(1-7) on pre-contracted tissues produced concentration-dependent relaxation, which was also inhibited by A-779. HOE-140, the selective antagonist of the bradykinin in B₂ receptor did not produce any significant effect on angiotensin II-induced relaxation. Pre-incubation of denuded-rings with N G-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced angiotensin II-induced relaxation. On the other hand, neither indomethacin nor tetraethylammonium (TEA) produced any significant effect. The major new finding of this work is that high concentrations of angiotensin II induce relaxation of the rat carotid via activation of the NO-cGMP pathway through a mechanism that seems to be partially dependent on activation of angiotensin-(1-7) receptors.     

Methionine proximity assay,a novel method for exploring peptide ligand-receptor interaction

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: receptor-bound angiotensin II adopts an extended structure

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.     

Molecular cloning, characterization, and distribution of the gerbil angiotensin II AT2 receptor

We isolated a cDNA clone encoding the gerbil AT2 receptor (gAT2) gene from a gerbil adrenal gland cDNA library. The full-length cDNA contains a 1,089-bp open reading frame encoding 363 amino acid residues with 90.9, 96.1, and 95.6% identity with the human (hAT2), rat (rAT2), and mouse AT2 (mAT2) receptors, respectively. There are at least seven nonconserved amino acids in the NH2-terminal domain and in positions Val196, Val217, and Met293, important for angiotensin (ANG) II but not for CGP-42112 binding. Displacement studies in adrenal sections revealed that affinity of the gAT2 receptor was 10-20 times lower for ANG II, ANG III, and PD-123319 than was affinity of the rAT2 receptor. The affinity of each receptor remained the same for CGP-42112. When transfected into COS-7 cells, the gAT2 receptor shows affinity for ANG II that is three times lower than that shown by the hAT2 receptor, whereas affinities for ANG III and the AT2 receptor ligands CGP-42112 and PD-123319 were similar. Autoradiography in sections of the gerbil head showed higher binding in muscles, retina, skin, and molars at embryonic day 19 than at 1 wk of age. In situ hybridization and emulsion autoradiography revealed that at embryonic day 19 the gAT2 receptor mRNA was highly localized to the base of the dental papilla of maxillary and mandibular molars. Our results suggest selective growth-related functions in late gestation and early postnatal periods for the gAT2 receptor and provide an essential basis for future mutagenesis studies to further define structural requirements for agonist binding.     

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.     

Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central alpha helix, a disordered N-terminal His1-Phe6 segment and a 3(10) Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2beta as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel beta sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.     

Renal Proteinase-activated Receptor 2, a New Actor in the Control of Blood Pressure and Plasma Potassium Level

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled membrane receptor that is activated upon cleavage of its extracellular N-terminal domain by trypsin and related proteases. PAR2 is expressed in kidney collecting ducts, a main site of control of Na⁺ and K⁺ homeostasis, but its function remains unknown. We evaluated whether and how PAR2 might control electrolyte transport in collecting ducts, and thereby participate in the regulation of blood pressure and plasma K⁺ concentration. PAR2 is expressed at the basolateral border of principal and intercalated cells of the collecting duct where it inhibits K⁺ secretion and stimulates Na⁺ reabsorption, respectively. Invalidation of PAR2 gene impairs the ability of the kidney to control Na⁺ and K⁺ balance and promotes hypotension and hypokalemia in response to Na⁺ and K⁺ depletion, respectively. This study not only reveals a new role of proteases in the control of blood pressure and plasma potassium level, but it also identifies a second membrane receptor, after angiotensin 2 receptor, that differentially controls sodium reabsorption and potassium secretion in the late distal tubule. Conversely to angiotensin 2 receptor, PAR2 is involved in the regulation of sodium and potassium balance in the context of either stimulation or nonstimulation of the renin/angiotensin/aldosterone system. Therefore PAR2 appears not only as a new actor of the aldosterone paradox, but also as an aldosterone-independent modulator of blood pressure and plasma potassium.