Solubilization and hydrodynamic characterization of guanine nucleotide-sensitive vasoactive intestinal peptide-receptor complexes from rat intestine

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Abstract
The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 .mu.M unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P] ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component. SDS.sbd.PAGE analysis revealed the presence in this peak of the Mr 42,000 .alpha. subunit of the Gs protein. Sucrose density gradient ultracentrifugation of the 5.2- and 3.1-nm components isolated on Sephacryl S-300 showed that they displayed apparent sedimentation coefficients of 6.7 and 3.9 S, respectively. From these results, the molecular weight of both components was estimated to be 152,000 and 54,000, respectively. It was concluded that intestinal VIP-receptor complexes of Mr 54,000 were solubilized in a major form of Mr 152,000 containing a Gs protein that was sensitive to GTP regulation.

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