Functional GHRH receptor carboxyl terminal isoforms in normal and dwarf (dw) rats

Abstract

GHRH plays a critical role in pituitary somatotroph development and function, actions which are mediated by a G-protein coupled receptor (GHRHr) that has been recently cloned. PCR amplification of rat pituitary mRNA using primers that span the GHRHr coding region resulted in two distinct products. When sequenced, the two isoforms were identical through bp 1278 of the GHRH coding region. However, the novel variant, which we have termed GHRHr , contains a 131 bp deletion (1279-1408) and resumes at bp 1409 in the 3 UTR of the previously identified transcript (GHRHr ). The identical isoforms were present in pituitaries from dwarf (dw) rats. The predicted amino acid sequence for the alternate receptor isoform differs from the published amino acid sequence at the extreme carboxyl terminus, with the last 5 amino acids of the published sequence replaced and an additional 17 amino acids added to the sequence. When translated in vitro or expressed as an epitope-tagged construct in non-GHRHr containing cell lines, the GHRHr mRNA produces a 42 kDa protein product, appropriately larger than the 40 kDa product of GHRHr mRNA. Furthermore, GHRHr retains the ability to promote cAMP generation in response to GHRH when expressed in non-GHRHr containing cell lines. These results indicate the presence of a splice variant of rat GHRHr mRNA present in normal and dw rat pituitary that codes for a functional receptor protein with an alternate carboxyl terminal domain. These findings raise the possibilities of target cell regulation of GHRH response, modulation of response through receptor isoform interactions and the involvement of multiple intracellular signaling pathways.

Increased lactotrophs despite decreased somatotrophs in the dwarf (dw/dw) rat: a defect in the regulation of lactotroph/somatotroph cell fate?

“…However, after taking into account the lower basal GH release and initial GH stores in the dw/dw cultures, the differences in GHRH responsiveness were less marked. The dw/dw somatotroph expresses a functional GHRH receptor, but at a lower abundance (Carmignac & Robinson 1990, Zeitler et al 1998, which could explain its lower sensitivity to GHRH. Comparable total numbers of lactotrophs were present in 5 day cultures.…”

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

“…Additional human GHRH receptor splice variants have been reported in several different cancers (Rekasi et al, 2000), and the detection of both GHRH and GHRH splice variants in human cancers raises the possibility that GHRH may exert an autocrine effect on regulation of neoplastic cell growth (Busto et al, 2002;Halmos et al, 2002). Alternative RNA processing also likely contributes to the carboxyl-terminal heterogeneity observed for the porcine GHRH receptor (Hsiung et al, 1993) and for the dwarf rat GHRH receptor (Zeitler et al, 1998). The two porcine receptor variants differ in their ability to bind GHRH and activate cAMP production in transfected cells (Hassan, 2001).…”

Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand–receptor interaction

“…In mammals, mRNA alternative splicing occurs at the third IL (Mayo 1992, Hashimoto et al 1995, McElvaine & Mayo 2006 or the C-terminal tail of GHRHR (Hashimoto et al 1995, Zeitler et al 1998. In chickens, alternative mRNA splicing of K7 M. The pGL3-CRE-luciferase reporter construct was co-transfected with cGHRHR or cGHRHR mutant expression plasmid into CHO cells to monitor the changes in intracellular cAMP levels after agonist treatment.…”

“…In mammals, GHRHR splice variants with altered signaling properties and ligand-binding affinity have been reported (Mayo 1992, Hashimoto et al 1995, Zeitler et al 1998, Hassan 2001. In rats, a GHRHR variant with an insertion of 41 amino acids in the third intracellular loop (IL3) could bind GHRH with high affinity, but fails to increase the intracellular cAMP levels (Mayo 1992, Miller et al 1999.…”

“…In contrast, another rat GHRHR variant with a substitution of the last five amino acids in the carboxyl terminus retains the ability to promote cAMP accumulation when tested in non-GHRHR expressing cells (Zeitler et al 1998). In humans, the retention of the intronic sequence generates a variant encoding a C-terminally truncated GHRHR with five transmembrane domains (Hashimoto et al 1995, McElvaine & Mayo 2006.…”