Hydropathicity analysis of 39 G-protein-coupled receptors (GPCR) reveals seven hydrophobic stretches corresponding to membrane spanning alpha-helices. The alignment of the primary sequences shows a high degree of homology in the GPCR transmembrane regions. 3D models of 39 GPCRs were generated using the refined model of bacteriorhodopsin as a template. Five cationic neurotransmitter receptors (serotonergic 5-HT2, dopaminergic D2, muscarinic m2, adrenergic alpha 2 and beta 2 receptors) were taken as prototypes and studied in detail. The 3D models of the cationic neurotransmitter receptors, together with their primary structure comparison, indicate that the agonist binding site is located near the extracellular face of the receptor and involves residues of the membrane-spanning helices 3, 4, 5, 6, and 7. The binding site consists of a negatively-charged Asp located at the middle of transmembrane helix 3 and a hydrophobic pocket containing conserved aromatic residues on helices 4, 5, 6, and 7. To define the precise receptor-ligand interactions, the natural neurotransmitters were docked into the binding sites. Residues responsible for the affinity, selectivity, and eventually stereospecificity of dopamine, adrenaline, noradrenaline, serotonin, and acetylcholine for their receptors were identified. The ligands are involved in electrostatic interactions as well as hydrogen bonds and specific hydrophobic aromatic interactions. All the GPCRs possess invariant hinge residues, which might be responsible for a conformational change during agonist binding and therefore influence dissociation and association of G-proteins to the receptors. The role of hydrophobic interactions and hydrogen bonds in the conformational change of the receptors, modulating the coupling to the G-protein, is discussed with regard to these residues. The models are in agreement with published data obtained from mutagenesis and labeling studies and represent important working hypotheses to direct future mutagenesis studies. They also enable structure-activity relationship studies and more rational drug design. The 3D models of other G-protein-coupled receptors have been generated in a similar way.
To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a threedimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-Å deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.The neurohypophysial hormones arginine vasopressin (AVP) 1 and oxytocin (OT) are two closely related nonapeptides that exhibit a high degree of functional diversity through interaction with specific receptors. Four different receptor subtypes have been characterized and possess different pharmacological and G protein coupling properties (1). V2 vasopressin receptors are coupled to adenylyl cyclase and are responsible for the antidiuretic effect of AVP. V1a and V1b vasopressin receptors are both coupled to phospholipase C. V1a receptors are involved in blood pressure control and in all other known functions of AVP, except for the stimulation of corticotropin secretion by the adenohypophysis which is mediated via V1b receptor subtype. OT receptors are coupled to phospholipase C and are responsible for the galactobolic and uterotonic effects. Recently, cDNAs for rat, human, and pig V2 receptors (2-4), rat and human V1a receptors (5, 6), pig and human OT receptors (4, 7), fish arginine vasotocin (AVT) receptor (8), and human V1b receptor (9) have been cloned and sequenced. This confirmed that these receptors belong to the G protein-coupled receptor (GPCR) family, characterized by seven hydrophobic putative ␣-helical transmembrane regions. All subtypes cloned so far are similar to each other in both identity and size. The level of conservation is remarkable, not only in the transmembrane domains but also in the first and second extracellular loops. Despite this high degree of receptor identity and a strong structural homology between the two hormones, the receptor subtypes are distinguished on the basis of different pharmacological and functional profiles. Many potent selective agonists as well as peptidic and nonpeptidic antagonists have been developed and used as pharmacological probes for the characterization of AVP and OT receptor subtypes (10).Previous molecular ...
Using a three-dimensional model of G protein-coupled receptors (GPCR), we have previously succeeded in docking the neurohypophysial hormone argininevasopressin (AVP) into the Vla receptor. According to this model, the hormone is completely embedded in the transmembrane part of the receptor. Only the side chain of the Arg residue at position 8 projects outside the transmembrane core of the receptor and possibly interacts with a Tyr residue located in the first extracellular loop at position 115. Residue 8 varies in the two natural neurohypophysial hormones, AVP and oxytocin (OT); similarly, different residues are present at position 115 in the different members of the AVP/ OT receptor family. Here we show that Arg8 is crucial for high affinity binding of AVP to the rat Vla receptor. Moreover, when Tyrl15 is replaced by an Asp and a Phe, the amino acids naturally occurring in the V2 and in the OT receptor subtypes, the agonist selectivity of the Vla receptor switches accordingly. Our results indicate that the interaction between peptide residue 8 and the receptor residue at position 115 is not only crucial for agonist high affinity binding but also for receptor selectivity.
Although the majority of the ion pairs found in proteins consists of two charges of opposite sign, the observation of some unusual arrangements of two arginines led us to a search of such occurrences in the Brookhaven Protein Data Bank. We have found 41 Arginine-Arginine interactions with a C zeta ... C zeta distance less than 5 A. Computer graphics analysis of these structures shows that most of the Arg-Arg pairs are found in the vicinity of the surface of the proteins, in an easily hydrated region. In order to determine which factors could stabilize such arrangements of species of similar charge, we have carried out AM1 semi-empirical calculations on a model of two guanidinium ions surrounded by several water molecules. The results show the existence of stable clusters with six or more water molecules, with distances between C zeta atoms around 3 A. The bridging role of the water molecules is an important structural and energetic feature and we find bridges of two and three molecules between the guanidinium ions. These results are in good agreement with the structures found in our search of the experimental data. Enhancement of the electrostatic potential around these clusters, when compared to one of the guanidinium ions alone, is also demonstrated.
We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.
The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D20 was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11 -36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N-terminus (residues 1-9) appears as an unstructured mobile segment.Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 f 0.6 pM at pH 2 -8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.Neuropeptide Y is a 36-amino-acid peptide widely distributed throughout both the central and peripheral nervous systems. Co-located with noradrenaline in many neurons of the sympathetic nervous system, it is thought to be involved in the control of the cardiovascular system (Lundberg et al., 1982;Everitt et al., 1984). In vivo administration results in a potent vasoconstriction which is independent of adrenergic mechanisms.Neuropeptide Y is a member of the pancreatic polypeptide family and, based on sequence analogy with avian pancreatic polypeptide for which an X-ray crystal analysis exists (Blundell et al., 1981), a similar tertiary structure has been proposed (Allen et al., 1987). This model predicts an amphiphilic a-helix between residues 14 -32 with strong hydrophobic interactions between one face of the a-helix and the amino-terminal polyproline-like helix, that also has some amphiphilic character.Recently, an NMR study of the secondary structure of neuropeptide Y (Saudek and Pelton, 1990) showed that the ular dynamics calculations to generate a three-dimensional model for this peptide. The tyrosine fluorescence intensity and anisotropy data in aqueous buffers and in the presence of acetonitrile confirms some structural and dynamic features of the dimer model, and provides values for the dimer dissociation constant as a function of pH. MATERIALS AND METHODS Peptide synthesisPorcine neuropeptide Y was synthesized by standard solidphase peptide synthetic methods using the t-butyl benzyloxycarbonyl/benzyl protection strategy, purified by gel filtration (Sephadex C-10) and preparative CI8 reverse-phase HPLC, and identity and purity confirmed by amino acid analysis and fast-atom-bombardment mass spectroscopy, as reported previously (Saudek and Pelton. 1990).C-terminal segment from residues 11 -36 folds into an amphiphilic a-helix while the N-terminal segment, containing Spectra three prolines in both cis and tram conformations assumes no regular structure. Many NOES were detected also betw...
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