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Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies. ISSN:1932-6203

Publisher Summary This chapter reviews the physiological and clinical studies of growth hormone (GH) and growth hormone releasing factor (GRF). GRF is the last of the classical hypothalamic hormones isolated. The isolation of GRF was only possible by the identification of peripheral GRF-secreting tumors. A major problem in isolating GRF was the minute amount of peptide presents in the hypothalamus, which, in addition, contained high concentrations of the GH release-inhibiting hormone, somatostatin. Its application to animal and human studies has opened a new horizon in GH physiology and pathophysiology. The pulsatile pattern of GH secretion in several mammalian species is accounted for by a dynamic interaction of hypothalamic somatostatin and GRF at the somatotroph. The increased GH secretion of the male rat is accounted for at least in part by increased percentage of somatotrophs, increased GH secretion per cell, and increased sensitivity to GRF. Studies with GRF in GH-deficient subjects have demonstrated that the majority of these patients do not suffer from a pituitary defect, but instead from a hypothalamic neurosecretory defect giving rise to GRF deficiency. Preliminary data demonstrate that GRF accelerates growth in GH-deficient children. GRF offers the prospect of restoring endogenous GH secretion to children and adults with GH deficiency. In addition, it appears that to restore pulsatile GH secretion GRF may be administered either in a pulsatile or continuous fashion. The tight regulation of GH by nutritional factors and the known metabolic effects of GH suggest that it is an important regulator of metabolism.