| 9015861 |
Grasso P: Functional reconstitution of detergent-solubilized bovine calf testis luteinizing hormone/chorionic gonadotropin receptor into phospholipid vesicles. Recept Signal Transduct. 1996;6(2):53-62.
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. |
2(0,0,0,2) |