I.A.1. Characterization of the gastrin-releasing peptide (GRP) receptor expressed in baculovirus (BV). The GRP receptor is expressed in low amounts in most tissues, and a BV-expression system might allow high expression. We expressed the murine GRP-R (mGRP-R) in SF9 cells using a recombinant BV and characterized it structurally and functionally. High levels of expression were obtained and the receptor was coupled to phospholipase C and to G proteins. The glycosylation was more immature than in native mGRP-R and the expressed BV-mGRP-R had 23% of the mature receptor's glycosylation. The ability of this system to express mGRP-R in increased amounts should be useful for obtaining receptor for antibodies, performing reconstitution studies and GRP-R modulation by agonists. I.B. Characterization of GI peptide receptor by the development of selective agonists or antagonists. I.B.1. Characterization of guinea pig chief cell CCK (a) receptors using a newly developed selective CCK (a) receptor agonist ligan. In many cells CCK(a) and CCK(b) receptors are both present however it is difficult to study them if CCK(A) and CCK(b) receptors, however, the CCK(a) receptors have not been able to be identified with existing ligands. We developed a selective, high affinity CCK (a) specific ligand. CCK (a) receptors were identified on chief cells with this ligand even though present in 1/100 the density of CCK(b) receptors. For the first time the relationship between binding and coupling for this cell could be examined with this ligand. I.B.2 Structural requirements for GRP-R and neuromedin B receptor (NMB- R) antagonists. The GRP-R and NMB-R mediate the actions of these two peptides in mammals. The physiology and pathophysiology of NMB remains unclear because in contrast tot he GRP-R no antagonists exist. In this study with Prof. D. Coy, Tulane University, we applied the 5 strategies which yielded GRP-R antagonists to NMB. Neither [desmet9]NMB amides, esters, alkylamides; 8-9 NMB analogues or D-amino acid NMB substituted analogues functional as antagonists. Only D-amino acid substituted substance P analogues or somatostatin analogues functional as weak antagonists. These results suggest that these two receptors have markedly different structure-function relationships and novel strategies will be needed to identify NMB-R antagonists.
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