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...

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G protein-coupled receptors represent the largest and most diverse superfamily of transmembrane receptors and mediate the effects of a wide variety of extracellular stimuli. It is now well established that, like single transmembrane tyrosine kinase receptors (2), these heptahelical receptors can form oligomeric complexes in the plasma membrane (3-13). Evidence for this includes biochemical, biophysical, and functional data (3, 6, 7). In addition to homodimerization, heterodimerization can occur with some structurally related receptors in this superfamily (8 -10, 12). This has been implicated in the effects on ligand binding and acute effects on cellular signaling (8,9,12). Formation of heterodimers of nonfunctional B1 and B2 ␥-aminobutyric acid receptors have been shown to be necessary for binding and complete functional activity of native ligands (10). Dimeric complexes of fully functional opioid receptors have been shown to alter their selectivity, displaying decreased affinities for highly selective agonists and enhanced affinities for nonselective agonists (9). Dimerization between somatostatin receptors has also been found to alter the pharmacology and signaling of individual receptors (12). However, little is known about the relevance of receptor dimerization to longer term effects, such as cell growth.Gastrin and cholecystokinin (CCK) 1 are gastrointestinal and neuronal peptides with important regulatory roles in the digestive tract and nervous system, including both acute and more chronic trophic effects (14 -16). These functions are mediated by two receptors, the type A and type B CCK receptors, both belonging to the class I family of G protein-coupled receptors and exhibiting 48% structural homology with each other (17, 18). These two receptors can be distinguished on the basis of their structural specificity, with both recognizing gastrin and CCK but with each having markedly distinct sensitivities to the state of tyrosine sulfation of those peptides.Although additional subtypes of CCK receptors have been predicted to exist, based on the complexity of the pharmacology and physiology of gastrin and CCK in vivo, only these two CCK receptor cDNAs have thus far been identified. It is possible that receptor oligomerization contributes to the pharmacology that has been observed and may even play a role in cell growth. We recently utilized bioluminescence resonance energy transfer (BRET) and immunoprecipitation to demonstrate that type A CCK receptors can exist as homodimers in living cells, with these complexes dissociated by agonist binding (1). To date there have been no reports of the ability of type B CCK receptors to dimerize or the possibility of the type A and B CCK receptors to form heterodimeric complexes.Indeed, using the same biophysical techniques, our current study demonstrated homodimerization of type B CCK receptors, as well as heterodimerization of this receptor with type A CCK receptors when co-expressed in cells. Of note, unlike the type A receptors (1), oligomerization of the type B CCK r...

Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs.