Identification of the Binding Sites of the SR49059 Nonpeptide Antagonist into the V1a Vasopressin Receptor Using Sulfydryl-reactive Ligands and Cysteine Mutants as Chemical Sensors

Abstract

To identify the binding site of the human V 1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or ␣-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V 1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radiolabeled antagonist [125 I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe 225 , located in transmembrane domain V, directly participates in the binding of the V 1a -selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family.The neurohypophysial antidiuretic hormone arginine vasopressin (AVP) 1 is involved in the regulation of body fluid osmolality, blood volume, and blood pressure via the stimulation of specific receptors currently classified into V 1a vascular (V 1a R) and V 2 renal (V 2 R) receptors. In addition, AVP modulates the adrenocorticotropic hormone secretion through V 1b pituitary (V 1b R) receptors. These different receptor subtypes along with the oxytocin receptor (OTR), which is classified in the same subfamily, possess distinct pharmacological profiles and intracellular second messengers (1, 2). Moreover, AVP belongs to the family of vasoactive and mitogenic peptides involved in physiological and pathological cell growth and differentiation (3). AVP has been shown to be one of the most powerful in vitro vasoconstrictor substances, and its vasoconstrictor and mitogenic actions may contribute to the pathogenesis of arterial hypertension, heart failure, and atherosclerosis (4, 5). AVP plays a role in the maintenance of blood pressure in several conditions, including upright posture, dehydration, hemorrhage, adrenal insufficiency, cardiac failure, and during surgery (6, 7). An abnormal vascular reactivity specific for AVP has been noted in models of genetic and experimental hypertension, and AVP is instrumental in the genesis and maintenance of several models of experimental hypertension (4, 5, 7). AVP...

Pharmacochaperones Post-translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-type and Mutant Vasopressin V2 Receptors

“…The energetically preferred conformations for SR49059 and SR121463 were selected from searches of their conformational spaces by the random search module in the Sybyl 6.9 package (TRIPOS Inc., St. Louis, MO). The starting orientation for docking of the V 1 R-specific antagonist SR49059 to the hV 2 R model was comparable to the reported SR49059-bound V 1A R model (17). In the case of SR121463, the initial orientation was derived after superposition of the common ring systems of SR121463 and SR49059.…”

(-)-7′-Isothiocyanato-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM841), a High-Affinity Electrophilic Ligand, Interacts Covalently with a Cysteine in Helix Six and Activates the CB1 Cannabinoid Receptor

“…Moreover, an improvement in affinity of nearly 2-fold was noted for ⌬ 9 -THC with C6.47(355)A, in which the WT cysteine is mutated to alanine, resulting in a more hydrophobic methyl group in this position (Fig. 5 (Zhu et al, 1996;Picone et al, 2002;Tahtaoui et al, 2003). Specific binding of [ 3 H]CP55940 was reduced from 303 Ϯ 58.4 pmol/g (buffer-treated) to 50.33 Ϯ 27.8 pmol/g (Fig.…”

“…Cysteine residues are the most likely candidates for reaction with isothiocyanates (Tahtaoui et al, 2003), which provided the rationale for the interactive docking studies to elucidate binding interactions for AM841. Of the 13 cysteines in human CB1, the two found in the E-2 loop and the two in the N terminus were excluded from consideration.…”

Vasopressin V1a and V1b Receptors: From Molecules to Physiological Systems

“…[From Thibonnier et al (494), with permission from American Society for Pharmacology and Experimental Therapeutics.] (F225) in the TM5 directly participates in the binding of the V1a receptor antagonist SR49059 (480). In the TM6, the mutation of three aromatic residues of the human V1a receptor, Trp-304 (W304), Phe-307 (F307), and Phe-308 (F308), reduced affinity for d(CH 2 ) 5 [Tyr(Me) 2 ]AVP and SR49059, but not for AVP binding, suggesting that these amino acids are necessary for agonist/antagonist discrimination (99).…”

“…The single or double amino acid substitutions of the human V1a receptor at I224V, I310V, G337A, G337A/I310V, G337A/ E324D, or G337A/I224V to the corresponding rat V1a receptor sequence dramatically increased the affinity of the rat V1a receptor antagonist OPC-21268, whereas the binding of AVP, as well as that of a peptide V1a receptor antagonist d(CH 2 ) 5 [Tyr(Me) 2 ]AVP (which later turned out to be a mixed V1a/OT receptor antagonist), and a selective nonpeptide V1a receptor antagonist SR49059, were minimally altered (494). Binding sites of SR49059 to the human V1a receptor, on the other hand, were analyzed using a sitedirected irreversible labeling strategy that combines mutagenesis of the receptor and binding of chemically reactive probes derived from SR49059 (480). This generates a chemical bond between the cysteine residue incorporated into the receptor and the sulfhydryl-reactive group in the SR49059 analog.…”