Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calpha methylation in molecular recognition. Application to substance P and the two neurokinin-1 receptor binding sites.

Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Distribution of substance P receptor (neurokinin-1 receptor) in normal ovine lung and during the progression of bronchopneumonia in sheep.

Substance P contributes to the physiological homeostasis of pulmonary airways and vasculature. During pneumonia, alterations in substance P production and receptor expression can influence bronchoconstriction and vascular perfusion. The distribution of substance P receptor [neurokinin-1 receptor (NK-1R)] in lungs of normal sheep and sheep with acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia caused by Mannheimia haemolytica was determined by immunohistochemistry (IHC). Three rabbit polyclonal antibodies generated to the same cytosolic C-terminal portion of NK-1R (residues 393-407) were tested. NK-1R immunoreactivity was traced in digital images and quantified with IPLAB software. There were no significant differences in NK-1R protein density between normal and infected lambs. Antibody 1 had the broadest distribution and intensity, and stained alveolar septae, smooth muscle cells of airways and vessels, epithelial cells of airways and alveoli, and submucosal glands. When all animals from the study were included, there was a trend towards decreased NK-1R immunoreactivity over time. The work suggests that (a) the density of NK-1R does not change during progression of bacterial (M. haemolytica) bronchopneumonia, (b) NK-1R is widely distributed in ovine lung and decreases with age, and (c) antibodies to the same NK-1R cytosolic region can vary in specificity and affinity.     

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10⁻⁶M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.     

The substance P/neurokinin-1 receptor system in lung cancer: Focus on the antitumor action of neurokinin-1 receptor antagonists

The last decades have seen no significant progress in extending the survival of lung cancer patients and there is an urgent need to improve current therapies. The substance P (SP)/neurokinin-1 receptor (NK-1R) system plays an important role in the development of cancer: SP and NK-1R antagonists respectively induce cell proliferation and inhibition in human cancer cell lines. No study of the involvement of this system in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells has been carried out in depth. Here, we demonstrate the involvement of the SP/NK-1R system in human H-69 (SCLC) and COR-L23 (NSCLC) cell lines: (1) they express isoforms of the NK-1R and mRNA for the NK-1R; (2) they overexpress the tachykinin 1 gene; (3) the NK-1R is involved in their viability; (4) SP induces their proliferation; (5) NK-1R antagonists (Aprepitant (Emend), L-733,060, L-732,138) inhibit the growth of both cell lines in a concentration-dependent manner; (6) the specific antitumor action of these antagonists against such cells occurs through the NK-1R; and (7) lung cancer cell death is due to apoptosis. We also demonstrate the presence of NK-1Rs and SP in all the human SCLC and NSCLC samples studied. Our findings indicate that the NK-1R may be a promising new target in the treatment of lung cancer and that NK-1R antagonists could be new candidate antitumor drugs in the treatment of SCLC and NSCLC.     

Role of Arg182 in the second extracellular loop of angiotensin II receptor AT2 in ligand binding.

The phenolic side chain of Tyr(4) present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, (125)I-Ang II and [(125)I-Sar(1)-Ile(8)]Ang II, as well as the AT2 receptor-specific peptidic ligand (125)I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of (125)I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor.     

Involvement of serotonin transporter extracellular loop 1 in serotonin binding and transport

Residues Tyr-110 through Gly-115 of serotonin transporter were replaced, one at a time, with cysteine. Of these mutants, only G113C retained full activity for transport, Q111C and N112C retained partial activity, but Y110C, G114C and G115C were inactive. Poor surface expression was at least partly responsible for the lack of transport by G114C and G115C. In membrane preparations, Y110C through G113C all bound a high affinity cocaine analog similarly to the wild type. Treatment with methanethiosulfonate reagents increased the transport activity of Q111C and N112C to essentially wild-type levels but had no measurable effect on the other mutants. The decreased activity of Q111C and N112C resulted from an increase in the K(M) for serotonin that was not accompanied by a decrease in serotonin binding affinity. Superfusion experiments indicated a defect in 5-HT exchange. Modification of the inserted cysteine residues reversed the increase in K(M) and the poor exchange, also with no effect on serotonin affinity. The results suggest that Gln-111 and Asn-112 are not required for substrate binding but participate in subsequent steps in the transport cycle.     

Involvement of serotonin transporter extracellular loop 1 in serotonin binding and transport

Residues Tyr-110 through Gly-115 of serotonin transporter were replaced, one at a time, with cysteine. Of these mutants, only G113C retained full activity for transport, Q111C and N112C retained partial activity, but Y110C, G114C and G115C were inactive. Poor surface expression was at least partly responsible for the lack of transport by G114C and G115C. In membrane preparations, Y110C through G113C all bound a high affinity cocaine analog similarly to the wild type. Treatment with methanethiosulfonate reagents increased the transport activity of Q111C and N112C to essentially wild-type levels but had no measurable effect on the other mutants. The decreased activity of Q111C and N112C resulted from an increase in the K_M for serotonin that was not accompanied by a decrease in serotonin binding affinity. Superfusion experiments indicated a defect in 5-HT exchange. Modification of the inserted cysteine residues reversed the increase in K_M and the poor exchange, also with no effect on serotonin affinity. The results suggest that Gln-111 and Asn-112 are not required for substrate binding but participate in subsequent steps in the transport cycle.     

The neurokinin-1 and neurokinin-2 receptor binding sites of MDL103,392 differ

Several small molecule non-peptide antagonists of the NK-1 and NK-2 receptors have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these non-peptide antagonists lies within the bundle created by transmembrane domains IV–VII of the receptor and differs from the binding sites of peptide agonists and antagonists. The current investigation uses site-directed mutagenesis of the NK-1 and NK-2 receptors to elucidate the amino acids that are important for binding and functional activity of the first potent dual NK-1/NK-2 antagonist MDL103,392. The amino acids found to be important for MDL103,392 binding to the NK-1 receptor are Gln-165, His-197, Leu-203, Ile-204, Phe-264, His-265 and Tyr-272. The amino acids found to be important for MDL103,392 binding to the NK-2 receptor are Gln-166, His-198, Tyr-266 and Tyr-289. While residues in transmembrane (TM) domains IV and V are important in both receptors (Gln-165/166 and His-197/198), residues in TM V and VI are more important for the NK-1 receptor and residues in TM VII play a more important role in the NK-2 receptor. These data are the first report of the analysis of the binding site of a dual tachykinin receptor antagonist and indicate that a single compound (MDL103,392) binds to each receptor in a different manner despite there being a high degree of homology in the transmembrane bundles. In addition, this is the first report in which a model for the binding of a non-peptide antagonist to the NK-2 receptor is proposed.     

The extracellular domain of the neurokinin-1 receptor is required for high-affinity binding of peptides.

The neurokinin-1 receptor binds neurokinin peptides with the potency order of substance P > substance K > neurokinin B. Elucidating the molecular basis of differential peptide selectivity will require the localization of the binding domain on the receptor. In the present report, mutagenesis and heterologous expression experiments reveal that a segment of the extracellular N-terminal sequence of the neurokinin-1 receptor is required for the high-affinity binding of substance P and related peptide agonists. Substitution of amino acid residues in the N-terminal region of the receptor affects the binding affinity of both intact peptides and a C-terminal substance P "analog", but not of a nonpeptide antagonist. Glycosylation of the receptor does not change the peptide binding affinity. In addition, substitution of the valine-97 residue in the rat neurokinin-1 receptor by a glutamate residue increases the binding affinity of neurokinin B but not substance P or substance K, suggesting that the second extracellular segment is involved in peptide selectivity. These results indicate that the extracellular domains of neurokinin-1 receptor play a critical role in peptide binding.     

Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.     

Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon

Nerve signal substances, such as the tachykinin substance P (SP), may be involved in the changes that occur in response to tendinopathy (tendinosis). It is previously known that the level of SP innervation within tendon tissue is limited, but results of experimental studies have suggested that SP may have stimulatory, angiogenetic and healing effects in injured tendons. Therefore, it would be of interest to know if there is a local SP-supply in tendon tissue. In the present study, the patterns of expression of SP and its preferred receptor, the neurokinin-1 receptor (NK-1 R), in normal and tendinosis human Achilles tendons were analyzed by use of both immunohistochemistry and in situ hybridization. We found that there was expression of SP mRNA in tenocytes, and that tenocytes showed expression of NK-1 R at protein as well as mRNA levels. The observations concerning both SP and NK-1 R were most evident for tenocytes in tendinosis tendons. Our findings suggest that SP is produced in tendinosis tendons, and furthermore that SP has marked effects on the tenocytes via the NK-1 R. It cannot be excluded that the SP effects are of importance concerning the processes of reorganization and healing that occur for tendon tissue in tendinosis. In conclusion, it appears as if SPergic autocrine/paracrine effects occur in tendon tissue during the processes of tendinosis, hitherto unknown effects for human tendons.     

Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

Involvement of substance P and the NK-1 receptor in human pathology

The peptide substance P (SP) shows a widespread distribution in both the central and peripheral nervous systems, but it is also present in cells not belonging to the nervous system (immune cells, liver, lung, placenta, etc.). SP is located in all body fluids, such as blood, cerebrospinal fluid, breast milk, etc. i.e. it is ubiquitous in human body. After binding to the neurokinin-1 (NK-1) receptor, SP regulates many pathophysiological functions in the central nervous system, such as emotional behavior, stress, depression, anxiety, emesis, vomiting, migraine, alcohol addiction, seizures and neurodegeneration. SP has been also implicated in pain, inflammation, hepatitis, hepatotoxicity, cholestasis, pruritus, myocarditis, bronchiolitis, abortus, bacteria and viral infection (e.g., HIV infection) and it plays an important role in cancer (e.g., tumor cell proliferation, antiapoptotic effects in tumor cells, angiogenesis, migration of tumor cells for invasion, infiltration and metastasis). This means that the SP/NK-1 receptor system is involved in the molecular bases of many human pathologies. Thus, knowledge of this system is the key for a better understanding and hence a better management of many human diseases. In this review, we update the involvement of the SP/NK-1 receptor system in the physiopathology of the above-mentioned pathologies and we suggest valuable future therapeutic interventions involving the use of NK-1 receptor antagonists, particularly in the treatment of emesis, depression, cancer, neural degeneration, inflammatory bowel disease, viral infection and pruritus, in which that system is upregulated.     

Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function

The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.     

Extracellular domains of the neurokinin-1 receptor: structural characterization and interactions with substance P

The technical difficulties associated with the structure determination of membrane proteins have limited the structural information available for the ligand binding to G-protein coupled receptors (GPCRs). Here, we describe a reductionist approach to GPCR structure determination in which the extracellular domains of the receptor are examined by high-resolution NMR in the presence of a membrane mimetic. The resulting structural features are then incorporated into a molecular model of the receptor, utilizing the x-ray structure of rhodopsin to generate the topological orientation of the transmembrane helices. The results of our study of the neurokinin-1 receptor (NK-1R) and its interactions with substance P (SP) are detailed here. The structure of the N-terminus, NK-1R(1-39), and of the third extracellular loop, NK-1R(264-290), in the presence of dodecylphosphocholine micelles is described. Our findings provide a structural basis for the interpretation of the results from other methods including mutagenesis, fluorescence, and photoaffinity labeling experiments, resulting in an experimentally based, high-resolution model of SP binding to NK-1R.     

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.