Receptor Cloning and Mutagenesis

Cloning of the MK-0677 receptor was achieved by Howard et al. (13) using cRNA pools derived from a swine pituitary cDNA library. Expression of the receptor in Xenopus oocytes was detected by measuring MK-0677 induced Ca2+ elevation. Given the low level of receptor expression in the pituitary, high sensitivity was required. It was obtained by co-injecting cRNA for the bioluminescent Ca2+ sensitive protein aequorin along with cRNA for the G-protein a-subunit Ga11. The initially cloned nucleotide sequence was used to obtain full length swine and human GHS receptor cDNAs. They encode polypeptides of 366 amino acids with seven transmembrane domains and with approx 93% identity comparing the swine and human receptors. They are novel receptors whose sequences are closest to those of neurotensin and TRH with approx 35% and 29% identity, respectively. Ligand binding Kj's were determined in displacement assays using [35S] MK-0677 bound to transiently trans-fected COS-7 cells and were in general agreement with ligand potencies in the rat pituitary cell assay: MK-0677 (Ki = 0.1 nM), GHRP-6 (K = 1.9 nM) and GHRP-2 (Ki = 0.21 nM) (13,29).

A functional receptor assay was established in HEK 293 cells based on Ca2+ elevation measured by aequorin bioluminescence. The assay, in which both MK-0677 and GHRP-6 are active, was used in mutagenesis studies to acquire some understanding of the receptor's essential functionality. An important structural feature of GH secreta-gogues is their basic amine. Presumably when a secretagogue binds, its amino group makes an electrostatic interaction with a negatively charged residue in the receptor. Attention focused on Glu124 in TM3 since it is in the approximate location of Asp113 in the ^-adrenergic receptor (67) and of Asp122 in the somatostatin type 2 receptor (68). In both instances these acidic residues, which are critical for receptor activation, are proposed to be amine binding sites. In fact, when the E124^Q124 mutant GHS human receptor was expressed in HEK 293 cells, both MK-0677 and GHRP-6 at 100 nM did not activate it as determined by the aequorin assay (69). The inference that E124 is an amine binding site in the GHS receptor and that this interaction is important for the activity of GHRP-6 and MK-0677 is a reasonable possibility assuming the E124^Q124 mutation did not cause a conformational change in the receptor.

0 0

Post a comment