The synthetic hexapeptide growth hormone releasing peptide 6 (GHRP-6) mediates growth hormone (GH) release from primary pituitary cells through a distinct mechanism from that controlled by growth hormone releasing hormone (GHRH) or somatostatin (1-3). Biochemical and pharmacological evidence supports the notion that GHRP-6 and the nonpeptide growth hormone secretagogs (GHSs) act through the same receptor. Numerous attempts to characterize the GHRP or GHS receptors (GHS-Rs) biochemically were frustrated by a low GHS-R abundance. The development of procedures for high-specific-activity [35S] radiolabeling of the nonpeptide GHS MK-0677 in conjunction with

From: Human Growth Hormone: Research and Clinical Practice Edited by: R. G. Smith and M. O. Thorner © Humana Press Inc., Totowa, NJ

Fig. 1. Expression cloning rationale. Schematic representation of GHS-R coupling to Ga11 and PLC leading to intracellular Ca2+ release, which can be measured with the photoprotein aequorin.

improved receptor preparation procedures led to the identification of a GHS-R binding site (4,5). The GHS-R bound [35S]-MK-0677 with high affinity, and the rank order of potency of diverse peptide and nonpeptide ligands for [35S]-MK-0677 displacement correlated with their in vivo GH secretory activity. Based on its binding characteristics the authors assumed that the GHS-R was a G protein-coupled receptor (GPC-R) found in low abundance in the anterior pituitary and hypothalamus. This data facilitated the development of a strategy to clone the GHS-R (Fig. 1). The assay for identification of the GHS-R relied on the knowledge that GHS-R activation leads to G protein-mediated activation of phosphoinositol-specific phospholipase C (PI-PLC) and subsequent calcium mobilization.

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