MOLECULAR ANALYSIS OF RAT PITUITARY AND HYPOTHALAMIC GROWTH-HORMONE SECRETAGOGUE RECEPTORS

GH release is thought to occur under the reciprocal regulation of two hypothalamic peptides, GH releasing hormone (GHRH) and somatostatin, v ia their engagement with specific cell surface receptors on the anteri or pituitary somatotroph. In addition; GH-releasing peptides, such as GHRP-6 and the nonpeptide mimetics, L-692,429 and MK-0677, stimulate G H release through their activation of a distinct receptor, the GH secr etagogue receptor (GHS-R). The recent cloning of the GHS-R from human and swine pituitary gland identifies yet a third G protein-coupled rec eptor (GPC-R) involved in the control of GH release and further suppor ts the existence of an undiscovered hormone that may activate this rec eptor. Using the human GHS-R as a probe, we report the isolation of a rat pituitary GHS-R cDNA derived from an unspliced, precursor mRNA. Th e rat cDNA encodes a protein of 364 amino acids containing seven trans membrane domains (7-TM) with >90% sequence identity to both the human and swine GHS-Rs. A single intron of similar to 2 kb divides the open reading frame into two exons encoding TM 1-5 and TM 6-7, thus placing the GHS-R into the intron-containing class of GPC-Rs. The intron maps to the site of sequence divergence between the human and swine type 1a and 1b GHS-R mRNAs. In addition, determination of the nucleotide sequ ence for the human GHS-R gene confirmed the position of an intron in t he human GHS-R gene at this position. A full-length contiguous cDNA fr om rat hypothalamus was isolated and shown to be identical in its nucl eotide and deduced amino acid sequence to the rat pituitary GHS-R. The cloned rat GHS-R binds S-35]MK-0677 with high affinity [dissociation constant (K-D) = 0.7 nM] and is functionally active when expressed in HEK-293 cells. Expression of the rat GHS-R was observed specifically i n the pituitary and hypothalamus when compared with control tissues.