Reconstitution of solubilized V1 vasopressin receptors of human platelets.
We describe the reconstitution of solubilized human platelet arginine vasopressin (AVP) receptors into phospholipid vesicles. Purified platelet plasma membranes enriched in AVP receptors [binding equilibrium dissociation constant (Kd) = 1.87 +/- 0.14 nM, maximum number of binding sites (Bmax) = 261 +/- 10 fmol/mg protein] were solubilized with 20 mM sodium cholate. Phospholipid vesicles made of 10% cholesterol, 20% egg phosphatidylcholine, and 70% egg phosphatidylserine were formed by bath sonication. Solubilized AVP receptors were incorporated into the vesicles while the detergent was removed by filtration through Sephadex G-100. The reconstituted receptors retained a high affinity for [3H]AVP (Kd = 3.19 +/- 0.13 nM, Bmax = 257 +/- 9 fmol/mg). Competition experiments with different AVP analogues confirmed the V1 vascular nature of the reconstituted receptors. Saturation experiments carried out with the agonist [3H]AVP and the V1 antagonist [3H]d(CH2)5Tyr(Me)AVP revealed that agonist binding to the reconstituted receptors was divalent cation dependent, whereas antagonist binding was not. Moreover, the affinity of the agonist [3H]AVP for the reconstituted receptors was modulated by the nonhydrolyzable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), whereas [3H]d(CH2)5Tyr(Me)AVP binding affinity was not. The phospholipid vesicles could be loaded with free fura-2 and displayed an enhanced fluorescence caused by calcium entry after addition of ionomycin. However, stimulation by AVP did not induce an increase of free calcium inside the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Thibonnier
M
M
Department of Medicine, University Hospitals of Cleveland, Ohio.
Bayer
A L
AL
Simonson
M S
MS
Snajdar
R M
RM
D001127:Arginine Vasopressin / Q000097:blood*; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D011945:Receptors, Angiotensin / Q000302:isolation & purification / Q000378:metabolism*; D017483:Receptors, Vasopressin; D012995:Solubility
D002412:Cations; D008081:Liposomes; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin