Activation of guanine nucleotide-binding protein (G protein)-coupled receptors is believed to involve conformational change that exposes a domain for G protein coupling at the cytosolic surface of the helical confluence, although the mechanisms for achieving this are not well understood. This conformational change can be achieved by docking a diverse variety of agonist ligands, known to occur by interacting with different regions of these receptors. In this study, we focus on the importance of a specific basic residue (Lys187) within the second extracellular loop of the receptor for the peptide hormone, cholecystokinin. Alanine-replacement and charge-reversal mutagenesis of this residue showed that it had no effect on the binding of natural peptide and nonpeptidyl ligands of this receptor but markedly interfered with agonist-stimulated signaling. It was demonstrated that this negative effect on biological activity could be eliminated with the truncation of the first 30 residues of the amino-terminal tail of this receptor. Complementary charge-reversal mutagenesis of each of the five conserved acidic residues within this region of the receptor in the presence of the charge-reversed Lys 187 revealed that only the Asp5 mutant fully reversed the negative functional impact of the Lys187 charge reversal. Thus, we have demonstrated that a basic residue within the second extracellular loop of the cholecystokinin receptor interacts with a specific acidic residue within the amino terminus of this receptor. This residue-residue interaction is nicely accommodated within a new molecular model of the agonist-occupied cholecystokinin receptor.
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