Solubilization of a large molecular weight form of the rat LHRH receptor

Abstract

The LHRH receptor has been solubilized from male rat anterior pituitary glands, using the zwitterionic detergent 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulphonate in the presence of a high concentration of sodium chloride. This method gave high yields (up to greater than 70%) of the LHRH-binding site from the membrane preparation. Ligand binding studies using LHRH analogues were carried out to determine dissociation constants for LHRH receptors both in situ in the membrane preparation and for solubilized LHRH receptors. For all the analogues the binding characteristics were similar in both preparations, suggesting that the solubilization procedure left the LHRH receptor undenatured. Gel filtration revealed an apparent molecular weight for the LHRH receptor of 100,000-160,000, with the mean value being approximately twice that found by others using sodium dodecyl sulphate-polyacrylamide gel electrophoretic techniques. The results indicate that the LHRH receptor probably exists in gonadotroph membranes as a large complex of more than one subunit.

Effects of epidermal growth factor and analogues of luteinizing hormone-releasing hormone and somatostatin on phosphorylation and dephosphorylation of tyrosine residues of specific protein substrates in various tumors.

“…(19,34), the first oncogene product found to contain phosphotyrosine and to be a substrate for EGF receptor. This protein is also the same size as a form of LH-RH receptor (35 These studies confirm that the extent of phosphorylation of individual tyrosine protein kinase substrates in the cell is controlled not only by specific tyrosine kinases, but also by specific tyrosine phosphatases. The phosphatases appear to have antitumor activity and can be stimulated by analogues of LH-RH and SS.…”

Regulation of luteinizing hormone-releasing hormone receptor binding by heterologous and autologous receptor-stimulated tyrosine phosphorylation.

“…The typical allosteric enzyme also functions as a complex of multiple subunits. Likewise, the LH-RHR appears to function as a polymer (29). These facts all support the similarity of the two systems.…”

“…Another possible mechanism capable of producing such regulation is activation of the receptor. The LH-RHR has been reported to be a 60-kDa protein (26)(27)(28) that can form a larger polymeric complex (29). One substrate of EGF-stimulated tyrosine kinase and RC-160-and [D-Trp6]LH-RH-stimulated tyrosine phosphatase is a 60-kDa protein (36).…”

Rhodopsin-family receptors associate with small G proteins to activate phospholipase D

“…PLD and PLC activities were monitored as production of [ 3 H]phosphatidylbutanol and [ 3 H]inositol phosphates, respectively 29 . Membrane binding was assayed as described 30 . This buffer contained 2 mM MgATP, 10 M NAD, 6 M digitonin, 30 mM butan-1-ol, and GTPgS, GPPCH 2 P or F − (NaF in the presence of 30 M BeCl 2 ) and BFA or C3 exoenzyme as required.…”