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-releasing hormone (GnRH) receptor represented an enhanced step in the experimental 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 systems for studies of signal transduction and desensitization, processes that may require immunologic detection of the receptor. Therefore, the GnRH receptor was tagged at its N-terminus using recombinant DNA procedures with the HA-1 epitope that is bound by a monoclonal antibody (12CA5). COS-1 cells expressing this receptor bound [(125)I]D-Ala6-desGly10-GnRH ethylamide (GnRH-A) with the expected high affinity (IC(50) = 0.47 nM), and were immunocytochemically stained by the 12CA5 antibody. Signal transduction was demonstrated by GnRH-induced [(3)H]inositol phosphate accumulation in receptor-expressing COS-1 cells. Western blotting of COS-1 cell membranes expressing the receptor revealed protein bands at 67, 57, and 32 kDa. Immunoprecipitation occurred when the solubilized receptor from COS-1 cell membranes was reacted with 12CA5 antibody and anti-mouse IgG Sepharose, and the presence of the receptor demonstrated either by its binding of [(125)I]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; [(32)P]i labeling of such cells during desensitization followed by immunoprecipitation of the N-epitope-tagged receptor was not associated with receptor phosphorylation. Finally, the epitope tagged receptor was expressed in the high-yield baculovirus/insect Sf9 cell system: the membrane receptor bound [(125)I]GnRH-A with slightly lowered affinity (IC(50) = 1.4 nM), and in Western blots yielded protein bands of 32, 56/57, 69, and 120/140 kDa. The development and validation of these heterologous systems will permit the study of several GnRH receptor-mediated processes that are poorly understood.
Neill
J D
JD
Department of Physiology and Biophysics, School of Medicine, University of Alabama at Birmingham, 35294, USA. Neill@uab.edu
Sellers
J C
JC
Musgrove
L C
LC
Duck
L W
LW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D015870:Gene Expression; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D011966:Receptors, LHRH / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology*; D013045:Species Specificity; D018411:Spodoptera; D014162:Transfection
D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007295:Inositol Phosphates; D012333:RNA, Messenger; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; D015544:Inositol 1,4,5-Trisphosphate