Oligomerization of the G protein-coupled cholecystokinin (CCK) receptor has been demonstrated, but its molecular basis and functional importance are not clear. We now examine contributions of transmembrane (TM) segments to oligomerization of this receptor using a peptide competitive inhibition strategy. Oligomerization of CCK receptors tagged at the carboxyl terminus with Renilla luciferase or yellow fluorescent protein was quantified using bioluminescence resonance energy transfer (BRET). Synthetic peptides representing TM I, II, V, VI, and VII of the CCK receptor were utilized as competitors. Of these, only TM VI and VII peptides disrupted receptor BRET. Control studies established that the β2-adrenergic receptor TM VI peptide that disrupts oligomerization of that receptor had no effect on CCK receptor BRET. Notably, disruption of CCK receptor oligomerization had no effect on agonist binding, biological activity, or receptor internalization. To gain insight into the face of TM VI contributing to oligomerization, we utilized analogous peptides with alanines in positions 315, 319, and 323 (interhelical face) or 317, 321, and 325 (external lipid-exposed face). The Ala 317,321,325 peptide eliminated the disruptive effect on CCK receptor BRET, whereas the other mutant peptide behaved like wild-type TM VI. This suggests that the lipid-exposed face of the CCK receptor TM VI most contributes to oligomerization and supports external contact dimerization of helical bundles, rather than domain-swapped dimerization. Fluorescent CCK receptor mutants with residues 317, 321, and 325 replaced with alanines were also prepared and failed to yield significant resonance transfer signals using either BRET or a morphological FRET assay, further supporting this interpretation.
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