Agonist-dependent Dissociation of Oligomeric Complexes of G Protein-coupled Cholecystokinin Receptors Demonstrated in Living Cells Using Bioluminescence Resonance Energy Transfer

Abstract

Dimerization of some G protein-coupled receptors has recently been demonstrated, but how widespread this phenomenon might be and its functional implicationsare not yet clear. We have utilized biophysical and biochemical techniques to evaluate whether the type A cholecystokinin (CCK) receptor can form oligomeric complexes in the plasma membrane and the impact of ligand binding and signaling on such complexes. We investigated the possibility of bioluminescence resonance energy transfer (BRET) between receptor constructs that included carboxyl-terminal tags of Renilla luciferase or yellow fluorescent protein. Indeed, co-expression of these constructs in COS cells resulted in the constitutive presence of a significant BRET signal above that in a series of controls, with this signal reduced by co-expression of competing non-tagged CCK receptors. The presence of an oligomeric complex of CCK receptor molecules was confirmed in co-immunoprecipitation experiments. Occupation of CCK receptors with agonist ligands (CCK or gastrin-4) resulted in the rapid reduction in BRET signal in contrast to the enhancement of such a signal reported after agonist occupation of the ␤ 2 -adrenergic receptor. These effects on CCK receptor oligomerization were concentration-dependent, correlating with the potencies of the agonists. A smaller effect was observed for a partial agonist, and no effect was observed for antagonist occupation of this receptor. Agonist-induced reduction in BRET signal was also observed for pairs of CCK receptors with a donor-acceptor pair situated in other positions within the receptor. Manipulation of the phosphorylation state of CCK receptor using protein kinase C activation with phorbol ester or inhibition with staurosporine had no effect on the basal level or agonist effect on CCK receptor oligomerization. This provides the first evidence for CCK receptor oligomerization in living cells, with insights that the active conformation of this receptor dissociates these complexes in a phosphorylation-independent manner.Guanine nucleotide-binding protein (G protein)-coupled receptors represent the largest superfamily of cell surface receptors, mediating the effects of a wide variety of extracellular stimuli. These have generally been assumed to initiate their signaling cascades upon agonist stimulation by interaction of a monomeric receptor molecule with a heterotrimeric G protein as a ternary complex (1, 2). However, increasing evidence suggests that at least some of these heptahelical membrane proteins can form dimers or oligomers in the plasma membrane under certain circumstances (3-9). The majority of such reports involve the biochemical or immunological demonstration of complexes migrating on a gel in a position too large to represent single receptors, often with demonstration of multiple epitope-tagged forms of the receptor in the complex. Such studies have been criticized for the possibility that the observed aggregation could represent an artifact of the solubilization or of handling concentrated preparations o...

The human serotonin1A receptor exhibits G-protein-dependent cell surface dynamics

“…Likewise, the presence of the bulky heterotrimeric G-protein complex associated with the receptor (since Gproteins, when bound to membrane receptors, could be considered as equivalent to cytoplasmic domains of membrane proteins) could further reduce (over the differences arising due to molecular mass of G-proteins) receptor diffusion, which would be partially relieved when the Gprotein dissociates from the receptor. Another possibility could be that the increase in receptor diffusion could reflect changes in the oligomeric state of the receptor, as has been shown for the δ-opioid receptor [41], and the cholecystokinin receptor [42] or their partitioning into or out of domains proposed to exist on the cell surface [7,9]. Incidentally, there is growing evidence on the compartmentalized localization of G-proteins in cholesterol-rich membrane domains [43] that have been reported to diffuse as separate entities on the membrane [44].…”

Secretin Receptor Oligomers Form Intracellularly during Maturation through Receptor Core Domains

“…These modifications were incorporated into a native human SecR open reading frame (ORF) that was subcloned through an EcoRV/SmaI blunt-end ligation from pBKCMV/SecR (28) into the cytomegalovirus-driven eukaryotic expression vector pcDNA3 (Invitrogen). The same mutations also were incorporated into Rlu and YFP carboxyl-terminally tagged versions of the same SecR coding sequence, minus the 5' and 3' untranslated regions (35). Construction of pcDNA3/SecR-CFP (27) and pcDNA3/CCKBR-YFP (36) were described previously.…”

“…Acceptor fluorescence was confirmed for all YFP-tagged mutants by direct excitation at 480 nm. BRET ratios (35) were calculated from baseline-corrected peak integrations as follows: [Em 510−590 -(Em 440−500 *Cf)]/Em 440−500 . The correction factor Cf = Em 510−590 /Em 440−500 defined the amount of signal in the acceptor portion of the spectrum that was attributable to donor bioluminescence, and was calculated separately for each experiment from cells expressing the appropriate Rlu-tagged SecR mutant alone.…”

“…The cells were maintained in a humidified chamber with 5% (v/v) CO 2 at 37 °C and were passaged twice per week on Corning (Acton, MA) tissue culture flasks. Cells inoculated to 10-cm Petri dishes were transfected according to a modified diethylaminoethyl (DEAE)-dextran method (35,37,38), and then were cultured for 24 h in DMEM with serum before lifting with 0.05% (w/v) trypsin and inoculating well plates or flasks for subsequent analyses. All transfections received the same amount of plasmid DNA regardless of the downstream experiment.…”

Molecular Basis of Agonist Binding to the Type A Cholecystokinin Receptor

“…This demonstrated that dimerization of the CCK receptor is not required for its biological activity, even though such dimers have been suggested to exist in bioluminescence resonance energy transfer studies (Cheng & Miller 2001). Of interest, CCK agonist binding to its receptor was shown to dissociate CCK receptor dimers in that study (Cheng & Miller 2001). The functional importance of CCK receptor oligomerization is unclear at the present time.…”