BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 939–945 (1996) ARTICLE NO. 1275 Solubilization and Characterization of a Growth Hormone Secretagogue Receptor from Porcine Anterior Pituitary Membranes Anna Pome ´s, 1 Sheng-Shung Pong, and James M. Schaeffer Cell Biochemistry and Physiology, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey, 07065 Received July 17, 1996 The discovery of a potential new GH therapy by small molecules that induce GH secretion (GHRP-6, L-692,429, MK-0677), has increased the interest in these GH secretagogues and their receptor and mecha- nism of action, which is different from the one of GHRH. We report the solubilization of the GH- secretagogue-receptor-ligand-G-protein complex (apparent molecular mass of approximately 255 kDa) from porcine anterior pituitary membranes using digitonin, after labelling the receptor with [ 35 S]MK-0677. The solubilized receptor showed high affinity (K D Å 122.2 { 14.4 pM) and low capacity (B max Å 3.8 { 0.9 fmol/mg protein). These values and the inhibition constants (K i ) for a series of GH secretagogues were similar to the values determined in membranes isolated from porcine anterior pituitary gland. The solubiliza- tion of the GH secretagogue receptor opens up the possibility for further molecular characterization and sequencing of the receptor protein, necessary step prior to the identification of the natural ligand that would act as a GHRH amplifying hormone, and that the GH secretagogues would mimic.  1996 Academic Press, Inc. 1. INTRODUCTION A new G-protein-linked receptor mediating the secretory response of the peptide (GHRP- 6) and peptidomimetic (MK-0677, L-692,429) GH secretagogues was recently identified [1]. These GH secretagogues do not interact with GHRH or somatostatin receptors [2,3]. They act at different levels to increase GH secretion in vivo and in vitro [2, 4-12]: a) they synergize with GHRH to produce a greater response than GHRH alone [2, 8, 9, 11, 12]; b) the GH secretagogues (see reference 1 for chemical structure) stimulate the release of GH from somato- trophs in the anterior pituitary gland [2, 10-12]; and c) they act on the arcuate nucleus in the hypothalamus to stimulate release of GHRH [13,14]. Their action is antagonized by the sub- stance P antagonist L-765,867 [1; Cheng, K. et al., unpublished]. The stimulatory action of GHRH and the GH secretagogues on GH release is mediated via different signal transduction pathways. GHRH stimulates the activity of adenylate cyclase increasing cAMP levels [2, 15]. However, other mechanisms of action have been proposed for the GH secretagogues (L- 692,429, MK-0677, GHRP-6): stimulation of GH secretion independently of cAMP accumula- tion in somatotrophs [2, 12], membrane depolarization and inhibition of potassium channels in rat somatotrophs [1], activation of protein kinase C [16] and increase phophatidylinositol hydrolysis [17], presumably with a consequent increase of intracellular free calcium levels. The GH secretagogues could mimic an unidentified endogenous hormone that potentiates the action of GHRH. A first step towards the identification of this hormone is the characterization of the receptor. In this study, we report the solubilization and characterization of this new receptor in order to have a better knowledge of its biochemical properties and the mechanism of action of GH secretagogues at the receptor level. In the solubilized state, it retains the properties that 1 Corresponding author. Fax:(908)-594-5700. E-mail: anna_pomes@Merck.Com. Abbreviations: GH, growth hormone; GTP-g-S, guanosine 5-O-(3-thiotriphosphate). 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 939