The availability of cloned GHS-Rs from several species allowed for a detailed comparison of their pharmacological properties in comparison with their naturally-occurring counterparts. Full-length cDNAs encoding swine, human, rat, mouse or dog GHS-R were placed into mammalian expression vectors and transiently or permanently expressed in monkey kidney cells (COS-7) or human embryonic kidney fibroblasts (HEK-293). Both cell types do not express detectable endogenous GHS-Rs. Binding and functional activation assays were utilized to characterize the cell lines and to compare the intrinsic potency of various GHS ligands. For the cloned GHS-Rs, two assays were utilized to measure receptor expression binding to cell membranes and aequorin bioluminescence in whole cells for a determination of the efficacy of functional coupling.
Characterization of the native GHS-R isolated from primary pituitary and hypothalamic tissue relied on pS-MK-0677 binding. In addition, GH release was measured in rat primary pituitary culture. All cloned GHS-Rs can be expressed at high levels in fibroblast recipient cell lines using both the radioligand and aequorin functional assays as a measure of GHS-R expression. This shows an example of an saturation isotherm for the cloned dog GHS-R, and a dose-response curve for functional activation of the cloned mouse GHS-R. Binding is saturable, of high affinity, identifying two classes of binding sites when expressed in heterologous cells. The identification of two classes of binding sites is most likely due to an excess of uncoupled receptors over available G proteins, leading to the presence of high and low affinity state GHS-Rs, as often observed when cloned receptors are overexpressed. This assertion is supported by the observation that only -50% of the binding can be blocked by GTPyS.
This compares the binding properties of the cloned GHS-Rs to the values of these same parameters obtained for GHS-R identified in tissue. In general, there is good agreement between intrinsic potency for binding and activation of the GHS-R in functional assays when compared across species for the cloned and native GHS-Rs. Site-directed mutagenesis of the human GHS-R has provided insights into key residues that are essential for GHS-R function. While overall amino acid sequence identity in the family of GPC-Rs is low, the seven transmembrane helices contain recognizable motifs which help to identify conserved structural elements that can be used in nucleating 2D GPC-R sequence alignments.
Diversity in the transmembrane region (especially the presence of a charge in the hydrophobic helices) can give clues to the location of amino acid residues that are potentially relevant for interaction with ligands. The location of essential amino acid residues of the GHS-R was initially based on a functional 2D sequence alignment of highly conserved motifs in related GPC-Rs and homology modehng based on the helical footprint of bacteriorhodopsin. In the 2D sequence alignment a comparison was made with the angiotensin-II, P2-2idrenergic, neurotensin, somatostatin-2 receptor and human, swine and rat GHS-Rs. Prior to mutagenesis, these data were combined to form a preliminary 3D GHS-R model docked with representative members of three classes of GHSs: the peptide, GHRP-6 and the non-peptides biphenyl benzolactam, L-692,585 and the spiroindolane, MK-0677. An important feature for GHS agonist bioactivity is the presence of a basic amino group. Based on conservation between human, swine and rat GHS-Rs and the 3D docking model, E124 in TM-3 was proposed to serve as the counter ion to the basic N found in GHS agonists such as MK-0677, L-692,585, and the amino terminus of GHRP-6.
An analogous residue is found in TM-3 of the P2 adrenergic receptor. When E124 is changed to Q, consistent with their basic side-chain, MK-0677, GHRP-6 and L-692,585 are no longer able to signal in the E to Q mutant. To confirm the role of this residue in receptor activation, L-168,740, an analog of MK-0677, was synthesized in which the primary amine side chain was modified to the corresponding alcohol which could then serve as the counterion to E124, though hydrogen bonding interactions. L-168,740 did not activate the wild type receptor at concentrations as high as 10 M. However, functional rescue of the E124Q mutant was achieved by L-168,740, with an EC50 of 1.3 LiM. Shown for comparison from the same experiment are dose-response curves for the activation of wild type and the E124Q mutant receptor by MK-0677, which gives a 200-fold decrease in potency against the E124Q. We conclude that the basic amine on the GHS agonist forms a salt bridge to the negative charge on the GHS-R. In addition, when the conservative change, E124 D was made, no effect on functional activation was observed, suggesting that a productive GHS-R confirmation was maintained.
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1. Significant species difference exists between sheep and rat
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