Comparison of the signalling properties of the long and short isoforms of the rat thyrotropin-releasing-hormone receptor following expression in rat 1 fibroblasts

Abstract

cDNA species encoding either the long or the short isoforms of the rat thyrotropin-releasing-hormone (TRH) receptor were expressed stably in Rat 1 fibroblasts, and clones expressing specific binding of [3H]TRH were detected and expanded. Clones expressing each of these receptors at levels up to 1 pmol/mg of membrane protein were selected for analysis. Reverse-transcriptase PCR on RNA isolated from these clones confirmed that each clone expressed only mRNA corresponding to the expected splice variant. Both receptor splice variants bound [3H]TRH with a Kd of some 80 nM when binding assays were performed in the presence of guanosine 5'-[beta gamma-imido]-triphosphate. In the presence of TRH, both receptor subtypes were able to cause stimulation of inositol phosphate generation in a pertussis-toxin-insensitive manner with similar EC50 values and to stimulate the mobilization of intracellular Ca2+, but, despite reports that TRH receptors can also interact with the G-proteins Gs and Gi2, neither receptor splice variant was able to modulate adenylate cyclase activity in either a positive or a negative manner. These data indicate that the long and short isoforms of the rat TRH receptor have similar affinities for TRH and display similar abilities to interact with the Gq-like G-proteins, but show no ability to regulate adenylate cyclase, at least when expressed in this genetic background.