Pharmacological Characterization

The pharmacological properties of type 1a and type 1b receptors were investigated using the aequorin and electrophysiological functional oocyte assays. In addition, GHS-R expression in COS-7 cells was characterized by [35S]-MK-0677 binding and by an aequorin assay conducted in transfected mammalian cells. Swine and human type 1a cRNAs expressed in oocytes responded to concentrations of MK-0677 ranging from 1 ^M to as low as 0.1 nM with an ED50 of ~5 nM. The amino-terminal truncated forms of the type 1a receptor (swine clone 7-3 and human 1146) were ~10-fold less active than their full-length counterparts (data not shown). Peptidyl and nonpeptidyl bioactive GHSs were active in a similar rank order of potency as observed for the native pituitary GHS-R. Type 1b cRNAs failed to give a response when injected into Xenopus oocytes or transfected into mammalian cells.

Binding experiments using [35S]-MK-0677 on crude cell membranes prepared from COS-7 transfectants confirmed that the type 1a cDNA, but not the type 1b cDNA, confers high-affinity, saturable, and specific binding (KD = 0.3 nM; Bmax = 0.2 pmol/mg cell membrane protein) of MK-0677 to a single class of noninteracting binding sites. Displacement of the radioligand by a variety of GHSs on the type 1a GHS-R was in strict correlation with their GH secretory activity. IC50s for MK-0677 and the peptide GHSs, GHRP-6, and GHRP-2 were 0.1, 1.9, and 0.21 nM, respectively. Other peptides, such as GHRH, CRF, GnRH, galanin, neurotensin, and neuromedin B, failed to show significant inhibition.

The functional significance of the truncated type 1b receptor cDNA is unknown. The type 1b GHS-R cDNA encodes the complete intracellular second and third loop responsible for G protein binding. Therefore, the type 1b cDNAs may be functional in the appropriate context. Naturally occurring examples of other truncated GPC-R mRNAs are rare and their function has not yet been elucidated. It is of interest to note that coexpression of artificially generated truncated muscarinic M2 or M3 receptors in COS-7 cells (TM1-5 and TM-6-7 expressed from two separate cDNAs) allows a functional muscarinic receptor to be reconstituted with ligand binding properties (and PI hydrolysis) as in the intact receptor (19). A similar study with mutant V2 vasopressin receptors was also recently reported (20). Given these results it is possible that a gene may exist that can restore functional activity of the type 1b GHS-R cDNA, encoding additional transmembrane domains. Alternatively, the predicted 24 C-terminal amino acids of the type 1b cDNA might encode a new TM-6 (H6) domain. The predicted amino-acid sequence is hydro-phobic and has similarity to helix 2 (H2) of the GHS-R. Helix 2 normally contacts H3, H6, H7, and H1. In the classical receptor folding model, H1 is positioned separate from the other six domains (TM2-7) and, therefore, may be open to additional contacts. One possible model for the type 1b GHS-R cDNA is that it indeed contains six transmembrane domains, H6 being similar to H2, allowing H6 to utilize contacts with H1 to restore a functional ligand-binding domain in this truncated GPC-R cDNA.

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