EPITOPE-TAGGED GONADOTROPIN-RELEASING-HORMONE RECEPTORS HETEROLOGOUSLY-EXPRESSED IN MAMMALIAN (COS-1) AND INSECT (SF9) CELLS

The molecular cloning and nucleotide sequencing of the gonadotropin-re leasing hormone (GnRH) receptor represented an enhanced step in the ex perimental effort to understand this key molecule in the reproductive process at a cell and molecular level. A subsequent step in this broad effort is heterologous expression of the receptor in model cell syste ms for studies of signal transduction and desensitization, processes t hat may require immunologic detection of the receptor. Therefore, the GnRH receptor was tagged at its N-terminus using recombinant DNA proce dures with the HA-1 epitope that is bound by a monoclonal antibody (12 CA5). COS-1 cells expressing this receptor bound [I-125]D-Ala(6)-desGl y(10)-GnRH ethylamide (GnRH-A) with the expected high affinity (IC50 = 0.47 nM), and were immunocytochemically stained by the 12CA5 antibody . Signal transduction was demonstrated by GnRH-induced [H-3]inositol p hosphate accumulation in receptor-expressing COS-1 cells. Western blot ting of COS-1 cell membranes expressing the receptor revealed protein bands at 67, 57, and 32 kDa. Immunoprecipitation occurred when the sol ubilized receptor from COS-1 cell membranes was reacted with 12CA5 ant ibody and anti-mouse IgG Sepharose, and the presence of the receptor d emonstrated either by its binding of [I-125]GnRH-A or by its detection on Western blots. Desensitization of inositol 1,4,5-trisphosphate (IP 3) production by N-epitope-tagged GnRH receptor expressing COS-1 cells was evoked by a five min GnRH pretreatment; [P-32](i) labeling of suc h cells during desensitization followed by immunoprecipitation of the N-epitope-tagged receptor was not associated with receptor phosphoryla tion. Finally, the epitope tagged receptor was expressed in the high-y ield baculovirus/insect Sf9 cell system: the membrane receptor bound [ I-125]GnRH-A with slightly lowered affinity (IC50 = 1.4 nM), and in We stern blots yielded protein bands of 32, 56/57, 69, and 120/140 kDa. T he development and validation of these heterologous systems Rill permi t the study oi several GnRH receptor-mediated processes that are poorl y understood. (C) 1997 Elsevier Science Ireland Ltd.