Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes.
An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents; D005640:Follicle Stimulating Hormone / Q000502:physiology; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007457:Iodine Radioisotopes; D008081:Liposomes / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000378:metabolism; D011092:Polyethylene Glycols; D011510:Proteolipids; D011962:Receptors, FSH / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D003902:Detergents; D007457:Iodine Radioisotopes; D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; D011962:Receptors, FSH; C020485:proteoliposomes; D015060:1,2-Dipalmitoylphosphatidylcholine; D006165:Guanylyl Imidodiphosphate; D011092:Polyethylene Glycols; D005640:Follicle Stimulating Hormone; D017830:Octoxynol; D002784:Cholesterol; D000262:Adenylyl Cyclases; C017251:1,2-oleoylphosphatidylcholine