Generation of polyclonal antiserum against the growth hormone secretagoguereceptor (GHS-R) - Evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has be en recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracell ular loop of the rat GHS-R. A competitive immunoassay showed that the antis erum had a specific affinity for the target peptide. To confirm the specifi city of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts f rom COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-t ype COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunos tained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kD a in the extracts from rat hypothalamus, pituitary and stomach by Western b lot analysis. These studies are the first to show the existence of GHS-R pr otein in the stomach. The antiserum for the GHS-R is sensitive and specific , and it would be useful for clarifying the roles of GHS/ghrelin. (C) 2001 Elsevier Science Inc. All rights reserved.