AJP - Endocrinology and Metabolism, Vol 259, Issue 5 E751-E756, Copyright © 1990 by American Physiological Society
Reconstitution of solubilized V1 vasopressin receptors of human platelets
M. Thibonnier, A. L. Bayer, M. S. Simonson and R. M. Snajdar
Department of Medicine, University Hospitals of Cleveland, Ohio.
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)
