The long isoform of the rat thyrotropin-releasing hormone receptor down-regulates Gq proteins.
A cDNA encoding the long isoform of the rat thyrotropin releasing hormone (TRH) receptor was expressed stably in HEK-293 cells. Polymerase chain reaction analysis confirmed expression of mRNA encoding only the long and not the short isoform. Activation of this receptor with TRH caused a large stimulation in production of inositol phosphates but did not produce either activation of basal or inhibition of forskolin-amplified adenylyl cyclase activity. Sustained exposure of these transfected cells to TRH resulted in a substantial reduction in cellular levels of Gq alpha-like immunoreactivity from some 12 to 5 pmol/mg of membrane protein without significant alterations in cellular levels of the alpha subunits of Gs, Gi1, Gi2, Gi3, or Go. Equivalent experiments in GH3 cells also indicated a marked down-regulation of Gq alpha/G11 alpha. Dose-response curves indicated that 20 nM TRH produced half-maximal down-regulation of cellular Gq-like immunoreactivity in the transfected HEK-293 cells and that half-maximal loss was produced within 3-4 h. Separation of the transfected HEK-293 cell membranes in SDS-polyacrylamide gel electrophoresis conditions able to resolve individual members of the Gq family of G-proteins demonstrated the presence of two related G-proteins. Both of the expressed Gq-like G-proteins were observed to be down-regulated in parallel by TRH. The similarity of dose-response curves and time-courses for loss of the two G-proteins indicates that the long isoform of the rat TRH receptor does not functionally select between these two transducer proteins. In GH3 cells both Gq alpha and G11 alpha, which are expressed at similar levels, were observed to be down-regulated equivalently by treatment with TRH.
Kim
G D
GD
Department of Biochemistry, University of Glasgow, United Kingdom.
Carr
I C
IC
Anderson
L A
LA
Zabavnik
J
J
Eidne
K A
KA
Milligan
G
G
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D007295:Inositol Phosphates; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases