Heterodimerization of Type A and B Cholecystokinin Receptors Enhance Signaling and Promote Cell Growth

Abstract

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...

Secretin Receptor Oligomers Form Intracellularly during Maturation through Receptor Core Domains

“…Figure 3A shows the BRET ratios calculated from a series of control experiments in which cells expressing differentially-tagged wild type SecRs yielded typical responses of 0.06 ± 0.01 that were well above the background responses achieved from those expressing Rlu-tagged receptors alone. These values represented about one-sixth of the signal achieved from an Rlu-YFP fusion protein (0.38 ± 0.01), and routinely were at least twice the signal obtained from cells coexpressing an Rlu-tagged SecR and a fully functional, YFP-tagged version of the structurally unrelated family A CCKBR (0.03 ± 0.01) (36). These important controls defined maximum and minimum BRET ratios that represented ideal and non-specific interactions, respectively, and thus the range within which signals such as those from wild type SecRs could be interpreted to indicate specific protein-protein interactions.…”

L’hétérodimérisation des récepteurs couplés aux protéines G

“…De plus, quelques études ont décrit l'implication de la dimérisation dans des aspects de la biologie des récepteurs comme leur biosynthèse, leur maturation, leur activation et leur régulation [7]. L'implication de la dimérisation dans des processus physiologiques comme la croissance cellulaire, ou physiopathologiques comme la vitréorétinopathie 1 , la pré-éclampsie, a également été rapportée [8]. Ce nouveau champ de recherche concernant les processus de dimérisation ouvre donc de nouvelles perspectives thérapeutiques.…”

Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor

“…Binding assays using the CCK-like radioligand 125 I-D-TyrGly-[(Nle 28,31 )CCK- 26 -33] were also performed, following the techniques established previously (31). These assays were performed in intact cells to establish cell surface expression and in membranes of select receptor constructs to determine the structural specificity of peptide binding.…”

“…The CCK-like peptide radioligand 125 I-D-Tyr-Gly-[(Nle 28,31 )CCK-26 -33] was synthesized, purified to homogeneity on reversed-phase HPLC, and radioiodinated in our laboratory as we described previously (30).…”

“…pcDNA3, respectively (31). Primers were designed to introduce mutations in transmembrane (TM) segments TM2 (position 2.61), TM3 (positions 3.28 and 3.29), TM6 (positions 6.51 and 6.52), or TM7 (position 7.39) of CCK1R and CCK2R constructs using the QuikChange TM site-directed mutagenesis kit (Stratagene, La Jolla, CA).…”