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.     

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.     

Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding

A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-?Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ?Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.     

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.     

Photoaffinity labeling demonstrates physical contact between vasoactive intestinal peptide and the N-terminal ectodomain of the human VPAC1 receptor

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide whose actions are mediated by VPAC receptors belonging to class II G protein-coupled receptors. To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67-137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109-120 fragment (GWTHLEPGPYPI). In a three-dimensional model of the receptor N-terminal ectodomain, this fragment is located on one edge of the putative VIP binding groove and encompasses several amino acids previously shown to be crucial for VIP binding (reviewed in Laburthe, M., Couvineau, A., and Marie, J. C. (2002) Receptors Channels 8, 137-153). Our data provide the first direct evidence for a physical contact between VIP and the N-terminal ectodomain of the hVPAC1 receptor.     

Des-aspartate-angiotensin I and angiotensin AT1 receptors in rat cardiac ventricles

The binding of 125I-[Sar1,Ile8]angiotensin II and 125I-angiotensin II to ventricular membranes of rat heart was studied. Displacement of bound 125I-[Sar1,Ile8]angiotensin II by its cold equivalents, angiotensin I, angiotensin II, angiotensin III, des-aspartate-angiotensin I, losartan, PD123319 and CGP42112B supports the presence of the AT1 and the near absence of the AT2 angiotensin receptor in adult rat ventricle. The presence of binding sites for des-aspartate-angiotensin I could account for its reported cardioprotective actions. Binding of 125I-angiotensin II but not that of 125I-[Sar1,Ile8]angiotensin II was partially displaced by GppNHp suggesting that a portion of the receptor population was in the active state with dissociated G-protein. Saturation experiments carried out in the absence and presence of 1 mM GppNHp showed similar magnitude of decrease in the number of receptors (Bmax from 26.2+/-1.3 to 15.7+/-1.1 fmol/mg protein) in [125I]-angiotensin II binding. However, the guanine nucleotide had no effect on the binding of 125I-[Sar1,Ile8]angiotensin II as has also been reported elsewhere, and may suggest that Sar1-Ile8-angiotensin II, being a partial agonist, binds to both the G-protein coupled and uncoupled states of the angiotensin receptors. The present study demonstrates that des-aspartate-angiotensin I binds to angiotensin receptors in the heart, and provides further evidence for its involvement in the pathophysiology of the organ.     

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.     

Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K(i) approximately 0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC(50) approximately 0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa(10),Tyr(14)]noc results in the irreversible labeling of a glycoprotein of approximately 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (approximately 6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of approximately 3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant receptor also yields a single labeled fragment of approximately 6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296-302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa(10)]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile(300) within the experimentally identified photoreactive region.     

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.     

Agonist-induced phosphorylation of the angiotensin II (AT(1A)) receptor requires generation of a conformation that is distinct from the inositol phosphate-signaling state

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT(1A)) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar(1),Ile(4),Ile(8)]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT(1A) receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT(1A) receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT(1) receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar(1),Ile(4),Ile(8)]AngII. Paradoxically, [Sar(1),Ile(4), Ile(8)]AngII produced a robust ( approximately 85% of AngII), dose-dependent phosphorylation of the wild type AT(1A) receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar(1),Ile(4),Ile(8)]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT(1A) receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.     

Diffuse pharmacophoric domains of vasoactive intestinal peptide (VIP) and further insights into the interaction of VIP with the N-terminal ectodomain of human VPAC1 receptor by photoaffinity labeling with [Bpa6]-VIP

The widespread 28-amino acid neuropeptide vasoactive intestinal peptide (VIP) exerts its many biological effects through interaction with serpentine class II G protein-coupled receptors named VPAC receptors. We previously provided evidence for a physical contact between the side chain at position 22 of VIP and the N-terminal ectodomain of the hVPAC1 receptor (Tan, Y. V., Couvineau, A., Van Rampelbergh, J., and Laburthe, M. (2003) J. Biol. Chem. 278, 36531-36536). We explored here the contact site between hVPAC1 receptor and the side chain at position 6 of VIP by photoaffinity labeling. The photoreactive para-benzoyl-l-Phe (Bpa) was substituted for Phe(6) in VIP resulting in [Bpa(6)]-VIP, which was shown to be a hVPAC1 receptor agonist in Chinese hamster ovary cells stably expressing the recombinant receptor. After obtaining the covalent (125)I-[Bpa(6)-VIP].hVPAC1 receptor complex, it was sequentially cleaved by cyanogen bromide, peptide N-glycosidase F, endopeptidase Glu-C, and trypsin, and the cleavage products were analyzed by electrophoresis. The data demonstrated that (125)I-[Bpa(6)-VIP] were covalently attached to the short 104-108 fragment within the N-terminal ectodomain of the receptor. The data were confirmed by creation of a receptor mutant with new CNBr cleavage site. In a three-dimensional model of the receptor N-terminal ectodomain, this fragment was located on one edge of the putative VIP-binding groove and was adjacent to the fragment covalently attached to the side chain at position 22 of VIP. Altogether these data showed that the central part of VIP, at least between Phe(6) and Tyr(22), interacts with the N-terminal ectodomain of the hVPAC1 receptor.     

Mutagenesis of the AT1 receptor reveals different binding modes of angiotensin II and [Sar1]-angiotensin II

Homology modeling of the structure of the AT1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT1 involves simultaneously all the receptor's residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys20, Arg23, Glu91 and Arg93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cell's response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar1]-angiotensin II suggests an important role for Arg23 of AT1 receptor in binding of the natural agonist. It is discussed whether some receptor's residues participate directly in the binding with AngII or whether they are part of a regulatory site.     

Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.

To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.     

Nephrectomy and a newly identified binding site for angiotensin II in the rat adrenal cortex.

Angiotensin II (Ang II) binding sites in adrenal glands of nephrectomized rats were investigated by in vitro autoradiography using 125I-[Sar1,Ile8]Ang II as ligands. Ang II binding site was increased to 161% in the cortex and decreased to 67% in the medulla 48 h after nephrectomy. In the medulla, the AT2 antagonist (PD123177, 5 microM) inhibited specific binding by 90% whereas the AT1 antagonist (DuP753, 5 microM) inhibited by only 10%. In contrast, in the cortex, neither DuP753 (5 microM) nor PD123177 (5 microM) substantially inhibited the binding. Binding in the presence of either the AT1 or AT2 antagonist was abolished by the simultaneous presence of both antagonists. These results suggest the presence of a new Ang II binding site with unique pharmacological properties and differing from currently known subtypes of Ang II receptors, in the adrenal cortex after nephrectomy.     

Formation of angiotensin-(1-7) from angiotensin II by the venom of Conus geographus

The binding of [3H]angiotensin II to AT(1) receptors on Chinese Hamster Ovary cells expressing the human AT(1) receptor (CHO-AT(1) cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT(1) receptor-selective antagonist [3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar(1),Ile(8)]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT(1) receptor. In this context, angiotensin-(1-7) is the most prominent fragment and angiotensin-(1-4) and angiotensin-(1-5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.     

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.     

Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)*neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.     

Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II

Previously, we showed that uterine arteries from late gestation pregnant ewes infused intravenously with angiotensin II (Ang II) for 24 h, displayed heightened responsiveness to Ang II in vitro. Furthermore, we found that a small population of ewes with a "preeclampsia-like" disorder also displayed this. Therefore, we have investigated the density and affinity of Ang II receptor subtypes in the uterine arteries from these groups. Ang II receptor binding was measured using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Uterine arteries from 24-h Ang II-infused ewes had a lower proportion of AT2 receptors (56.2+/-2.3%) than control (saline-infused) ewes (84.1+/-1.0%; P<0.05). The density of AT2 receptors was reduced (P<0.05) while the density of AT1 receptors was not different. Thus, 24-h infusions of Ang II selectively down-regulated AT2 receptors in the uterine artery, resulting in heightened Ang II reactivity. By contrast, the binding properties of Ang II receptor subtypes in uterine arteries from ewes with the "preeclampsia-like" disorder were not different from control ewes.