Functional reconstitution of detergent-solubilized bovine calf testis luteinizing hormone/chorionic gonadotropin receptor into phospholipid vesicles.
An LH/CG receptor-enriched fraction was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testicular homogenates and solubilized with Triton X-100. To confirm the functional nature of the detergent-solubilized LH/CG receptor, the extract was first incorporated by lipid hydration into phospholipid vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. LH/CG receptor incorporation was then determined by measurement of specific binding of [125I]hCG. Specific binding of [125I]hCG by the reconstituted receptor was saturable, time-dependent, and thermally stable at room temperature. Scatchard analysis of competitive binding data indicated the presence of a single class of high-affinity (6.9 x 10(10)M-1), low-capacity (17.5 fmol hCG/mg protein) binding sites. The reconstituted receptor was functionally coupled to adenylyl cyclase and responded to both LH and NaF with increased cyclic AMP (cAMP) production. Stimulation of LH/CG receptor-enriched proteoliposomes with LH resulted in concentration-dependent uptake of external calcium (as 45Ca2+), which was hormone-specific, saturable, and sensitive to blockade by voltage-dependent and voltage-independent calcium channel antagonists. Similar uptake could not be induced by sodium fluoride, (Bu)2 cAMP, GTP-gamma-S, cholera toxin, or pertussis toxin. These results indicate that the reconstituted LH/CG receptor, as is the membrane-associated receptor, is functionally coupled to signal transduction pathways involving both adenylyl cyclase activation and calcium mobilization, and is a reliable working model that will facilitate further examination of the molecular mechanisms of LH action.
Grasso
P
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Department of Biochemistry and Molecular Biology, Albany Medical College, NY 12208, USA.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002417:Cattle; D002648:Child; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D007986:Luteinizing Hormone / Q000378:metabolism*; D008297:Male; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D011863:Radioimmunoassay; D011974:Receptors, LH / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013737:Testis / Q000378:metabolism*
D002121:Calcium Channel Blockers; D006063:Chorionic Gonadotropin; D003902:Detergents; D008081:Liposomes; D011510:Proteolipids; D011974:Receptors, LH; C020485:proteoliposomes; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D017830:Octoxynol; D000262:Adenylyl Cyclases; D002118:Calcium