Structural and functional divergence of growth hormone-releasing hormone receptors in early sarcopterygians: lungfish and Xenopus.

The evolutionary trajectories of growth hormone-releasing hormone (GHRH) receptor remain enigmatic since the discovery of physiologically functional GHRH-GHRH receptor (GHRHR) in non-mammalian vertebrates in 2007. Interestingly, subsequent studies have described the identification of a GHRHR(2) in chicken in addition to the GHRHR and the closely related paralogous receptor, PACAP-related peptide (PRP) receptor (PRPR). In this article, we provide information, for the first time, on the GHRHR in sarcopterygian fish and amphibians by the cloning and characterization of GHRHRs from lungfish (P. dolloi) and X. laevis. Sequence alignment and phylogenetic analyses demonstrated structural resemblance of lungfish GHRHR to their mammalian orthologs, while the X. laevis GHRHR showed the highest homology to GHRHR(2) in zebrafish and chicken. Functionally, lungfish GHRHR displayed high affinity towards GHRH in triggering intracellular cAMP and calcium accumulation, while X. laevis GHRHR(2) was able to react with both endogenous GHRH and PRP. Tissue distribution analyses showed that both lungfish GHRHR and X. laevis GHRHR(2) had the highest expression in brain, and interestingly, X. laevis(GHRHR2) also had high abundance in the reproductive organs. These findings, together with previous reports, suggest that early in the Sarcopterygii lineage, GHRHR and PRPR have already established diverged and specific affinities towards their cognate ligands. GHRHR(2), which has only been found in xenopus, zebrafish and chicken hitherto, accommodates both GHRH and PRP.

Growth hormone-releasing factor mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary.

Porcine somatotropes can be separated by Percoll density gradient centrifugation into low (LD) and high density (HD) subpopulations that differ ultrastructurally and functionally. Here, we report the effects of growth hormone-releasing factor (GRF) on the cytosolic free calcium concentration ([Ca2+]i) of single LD and HD somatotropes. Resting [Ca2+]i in LD somatotropes was 2-fold higher than in HD cells. GRF induced [Ca2+]i increases in a similar percentage of somatotropes from both subsets. However, amplitude and kinetics of the responses were markedly different. In all responsive LD somatotropes, GRF evoked a rapid initial peak followed by a sustained plateau (plateau-type response). Blockade of extracellular Ca2+ entry by 3 mM EDTA, 2 mM CoCl2, or 100 microM verapamil completely abolished the plateau phase without affecting the initial Ca2+ spike. Conversely, only the plateau phase was preserved in thapsigargin (TG)-treated LD cells. The vast majority of GRF-responsive HD somatotropes exhibited a transient [Ca2+]i peak that returned gradually to baseline (transient-type response). This response was completely blocked by removal of extracellular Ca2+, whereas TG treatment had no effect. Taken together, our results indicate that the response of LD somatotropes to GRF depends on mobilization of Ca2+ of both extra- and intracellular origin, whereas that of HD somatotropes seems to be exclusively dependent on extracellular Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC). These findings are the first to demonstrate a differential effect of GRF on Ca2+ mobilization in two somatotrope subpopulations, and suggest the existence of differences in the GRF receptor(s) expressed in each subpopulation and/or in the intracellular signalling pathways activated upon GRF binding.

Identification of a pituitary growth hormone-releasing peptide (GHRP) receptor subtype by photoaffinity labeling.

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 Mr of 57,000 in human, bovine and porcine anterior pituitary membranes. Hexarelin and the spiroindoline sulfonamide MK-0677 displaced the Mr-57,000 photolabeled band with an apparent ED50 of 6x10(-7) M and 2x10(-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.

Binding sites for growth hormone-releasing peptide.

Growth hormone-releasing peptides (GHRPs) are known to release growth hormone (GH) in vivo and in vitro by a direct action on receptors in anterior pituitary cells. Measurement of second messengers released following somatotroph stimulation suggests the existence of more than one GHRP receptor subtype in the hypothalamic-pituitary system. Furthermore, hexarelin, a hexapeptide of the GHRP family and a potent GH secretagogue, is reported to increase left ventricular ejection fraction, suggesting the expression of specific myocardial GHRP binding sites. In order to confirm such a hypothesis, a photoactivatable derivative of hexarelin, Tyr-p-benzoyl phenylalanine-Ala-hexarelin, was developed. A putative GHRP receptor with an apparent relative molecular mass of 57,000 was specifically labelled and characterized in human, bovine and porcine anterior pituitary membranes using this hexarelin derivative. The existence of myocardial binding sites was also demonstrated using the same approach. The differential binding affinity of GHRP analogues to cardiac tissue raises the possibility of the existence of distinct GHRP receptor subtypes in the pituitary and the cardiovascular system, for which physiological roles have yet to be determined.