title: Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney.
abstract: Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.
authors.affiliation: Department of Molecular Pharmacology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101.
authors.forename: N
authors.initials: N
authors.lastname: Aiyar
authors.forename: W
authors.initials: W
authors.lastname: Valinski
authors.forename: P
authors.initials: P
authors.lastname: Nambi
authors.forename: M
authors.initials: M
authors.lastname: Minnich
authors.forename: F L
authors.initials: FL
authors.lastname: Stassen
authors.forename: S T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism; D001667:Binding, Competitive; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007679:Kidney Medulla / Q000032:analysis* / Q000378:metabolism; D008236:Lypressin / Q000378:metabolism; D008244:Lysophosphatidylcholines; D011945:Receptors, Angiotensin / Q000302:isolation & purification* / Q000378:metabolism; D017483:Receptors, Vasopressin; D012995:Solubility; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D006150:Guanine Nucleotides; D008244:Lysophosphatidylcholines; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D013873:Thionucleotides; D001127:Arginine Vasopressin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008236:Lypressin; D006160:Guanosine Triphosphate