SOLUBILIZATION OF A GUANINE NUCLEOTIDE-SENSITIVE PARATHYROID HORMONE-RECEPTOR COMPLEX FROM CANINE RENAL-CORTEX

Canine renal cortical PTH receptors were solubilized after occupancy of membrane-associated receptors with the agonist ligand [125I]bovine (b) PTH-(1-34). Stabilization of binding during solubilization required the use of high concentrations of BSA (optimally 5%) and appropriate detergents (0.5% 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate, 0.5% 3-[(3-cholamidopropanyl)dimethylammonio]1-propanesulfonate, or 0.5-1.0% digitonin). The soluble fraction (240,000 .times. gav supernatant) contained [125I]bPTH-(1-34) associated with macromolecular components as well as unbound [125I]bPTH-(1-34) that dissociated during solubilization. The soluble macromolecular complex had functional properties expected of a ternary complex consisting of [125I]bPTH-(1-34) receptor stimulatory guanine nucleotide-binding protein (N8). Thus, the dissociation of labeled PTH at 30 C was slow (t1/2 = 75 min); in the presence of GTP (10-4 M), 75% of the sites displayed rapid dissociation kinetics (t1/2 - 2.3 min). This effect was nucleotide specific, with GTP .simeq. GTP.gamma.S .simeq. GDP > GDP.beta.S > ITP .simeq. guanylylimidodiphosphate .mchgt. GMP .simeq. App(NH)p. ATP was ineffective. GTP produced a half-maximal response at a concentration of 200 nM. These results are consistent with the reported nucleotide specificity and affinity of purified N8. Treatment of membranes with N-ethylmaleimide during the binding reaction rendered the solubilized complex refractory to GTP. Gel filtration chromatography (Sepharose 6B) revealed a GTP-sensitive complex that eluted in the position expected of a detergent-free spherical protein of 180,000 daltons. This complex may consist of the 60,000 to 70,000-dalton PTH-binding subunit (previously identified by photoaffinity labeling) together with N8.