A peptide agonist acts by occupation of a monomeric G protein-coupled receptor: Dual sites of covalent attachment to domains near TM1 and TM7 of the same molecule make biologically significant domain-swapped dimerization unlikely

Membrane receptor dimerization is a well-established event for initiation o f signaling at growth factor receptors and has been postulated to exist for G protein-coupled receptors, based on correction of nonfunctional truncate d, mutant, or chimeric constructs by coexpression of appropriate normal com plementary receptor domains. In this work, we have directly explored the mo lecular composition of the minimal functional unit of an agonist ligand and the wildtype G protein-coupled cholecystokinin (CCK) receptor, using photo affinity labeling with a CCK analogue probe incorporating dual photolabile benzoylphenylalanine (Bpa) residues as sites of covalent attachment. This p robe, I-125-D-Tyr-Gly-[(Nle(28,31), Bpa(29,33))CCK-26-33], was shown to rep resent a full agonist and to specifically label the CCK receptor. Like prob es incorporating individual photolabile residues in these positions,(1,2) t he two Bpa residues in the dual photoprobe covalently labeled receptor doma ins in the amino-terminal tail outside TMI and in the third extracellular l oop outside TM7. Absence of demonstrable receptor dimerization after the es tablishment of dual sites of covalent attachment supports the presence of t hese two domains within a single receptor molecule. Demonstration of the co valent adduct of a single probe molecule with the two cyanogen bromide frag ments of the CCK receptor representing the expected domains further support s this interpretation. Thus, while domain-swapped dimerization of G protein -coupled receptors may be possible as a mechanism of rescue for nonfunction al molecules, it is not necessary for ligand binding and initiation of sign aling at a wild-type receptor in this superfamily. The functional unit for CCK action is normally a ligand-receptor monomer.