Identification of a Pituitary Growth Hormone-Releasing Peptide (GHRP) Receptor Subtype by Photoaffinity Labeling

Abstract

Hexarelin, an analogue of GHRP-6, in which D-Tryptophan has been replaced by its 2-methyl derivative, is known to release growth hormone (GH) in vivo and in vitro by direct action on receptors present in anterior pituitary cells. Measurement of second messengers (c-AMP, Ca⁺⁺, IP₃) upon somatotrophs stimulation, suggests the existence of more than one GHRP receptor subtype. In order to document such an hypothesis, we have used a new photoactivatable derivative of Hexarelin, Tyr-Bpa-Ala-Hexarelin. This derivative was shown to be fully active in the release of GH in vivo with neonate rats. Using this photoactivatable ligand, we have specifically labeled a protein with an apparent M_r of 57 000 in human, bovine and porcine anterior pituitary membranes. Hexarelin and the spiroindoline sulfonamide MK-0677 displaced the M_r −57 000 photolabeled band with an apparent ED₅₀ of 6×10⁻⁷ M and 2×10⁻⁵ M respectively. Taking into account the high efficiency (>60%) of covalent incorporation of the Bpa residue, this photoactivatable Hexarelin derivative has allowed the identification of a pituitary GHRP receptor subtype, which is apparently distinct from the recently cloned GH secretagogue receptor.