Interaction of delta-opioid receptors with multiple G proteins: a non- relationship between agonist potency to inhibit adenylyl cyclase and to activate G proteins

PL Prather, HH Loh and PY Law

Department of Pharmacology, School of Medicine, University of Minnesota, Minneapolis 55455.

The purpose of the present investigation was to determine whether the coupling of delta-opioid receptors to multiple G proteins in NG108-15 neuroblastoma x glioma cells is a characteristic limited to only this cell line (because of the high density of delta-opioid receptors) and to ascertain whether there is any correlation between delta-opioid agonist potency to inhibit adenylyl cyclase and to activate G proteins. Interactions between receptors and G proteins were investigated using agonist-stimulated incorporation of the photoreactive GTP analog azidoanilido[alpha-32P]GTP ([alpha-32P]AA-GTP) into G protein alpha subunits, with subsequent separation by urea/sodium dodecyl sulfate- polyacrylamide gel electrophoresis. In NG108-15, NS20Y, and N1E115 cell membranes, four alpha subunits (Gi2 alpha, one isoform of Gi3 alpha, and both isoforms of Go alpha) in the 39-41-kDa region were labeled with [alpha-32P]AA-GTP. The delta-opioid agonist [D-Ala2,D-Leu5]- enkephalin (DADLE) produced a dose-dependent, naloxone-reversible increase of [alpha-32P]AA-GTP incorporation into all four alpha subunit subtypes, in all cell lines tested. In addition, with the single exception of Gi3 alpha in NG108-15 cells, the maximal increases in incorporation of the photoaffinity label into all G alpha subunits induced by DADLE were similar. The Bmax values determined for delta- opioid receptors in NG108-15, NS20Y, and N1E115 cell membranes were 570, 370, and 120 fmol/mg of protein, respectively. Finally, although the IC50 values to inhibit intracellular cAMP production and affinity for DADLE were similar across the three cell lines, the EC50 values to produce labeling of the G alpha subunits between cell lines differed by > 100-fold. In fact, only in NS20Y cells were the IC50 and ED50 values comparable. Firstly, these results suggest that simultaneous coupling of the delta-opioid receptor to multiple G protein alpha subunits occurs in a variety of cell lines that express a range of receptor densities. Secondly, the magnitudes with which delta-opioid receptors interact with available G alpha subunits in response to agonist are approximately the same. Finally, there appears to be no relationship between the potency of agonists to inhibit adenylyl cyclase and that required for activation of G proteins.

Volume 45, Issue 5, pp. 997-1003, 05/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				P. A. Claude-Geppert, J. Liu, J. Solberg, L. J. Erickson-Herbrandson, H. H. Loh, and P.-Y. Law Antagonist Efficacy in MORS196L Mutant Is Affected by the Interaction between Transmembrane Domains of the Opioid Receptor J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 216 - 226. [Abstract] [Full Text] [PDF]

				I. D. Alves, K. A. Ciano, V. Boguslavski, E. Varga, Z. Salamon, H. I. Yamamura, V. J. Hruby, and G. Tollin Selectivity, Cooperativity, and Reciprocity in the Interactions between the {delta}-Opioid Receptor, Its Ligands, and G-proteins J. Biol. Chem., October 22, 2004; 279(43): 44673 - 44682. [Abstract] [Full Text] [PDF]

				I. Gomes, A. Gupta, J. Filipovska, H. H. Szeto, J. E. Pintar, and L. A. Devi A role for heterodimerization of {micro} and {delta} opiate receptors in enhancing morphine analgesia PNAS, April 6, 2004; 101(14): 5135 - 5139. [Abstract] [Full Text] [PDF]

				I. D. Alves, Z. Salamon, E. Varga, H. I. Yamamura, G. Tollin, and V. J. Hruby Direct Observation of G-protein Binding to the Human {delta}-Opioid Receptor Using Plasmon-Waveguide Resonance Spectroscopy J. Biol. Chem., December 5, 2003; 278(49): 48890 - 48897. [Abstract] [Full Text] [PDF]

				A. C. Charles, N. Mostovskaya, K. Asas, C. J. Evans, M. L. Dankovich, and T. G. Hales Coexpression of delta -Opioid Receptors with {micro} Receptors in GH3 Cells Changes the Functional Response to {micro} Agonists from Inhibitory to Excitatory Mol. Pharmacol., January 1, 2003; 63(1): 89 - 95. [Abstract] [Full Text] [PDF]

				N. A. Martin, M. T. Terruso, and P. L. Prather Agonist Activity of the delta -Antagonists TIPP and TIPP-psi in Cellular Models Expressing Endogenous or Transfected delta -Opioid Receptors J. Pharmacol. Exp. Ther., July 1, 2001; 298(1): 240 - 248. [Abstract] [Full Text]

				P. L. Prather, L. Song, E. T. Piros, P. Y. Law, and T. G. Hales delta -Opioid Receptors Are More Efficiently Coupled to Adenylyl Cyclase Than to L-Type Ca2+ Channels in Transfected Rat Pituitary Cells J. Pharmacol. Exp. Ther., November 1, 2000; 295(2): 552 - 562. [Abstract] [Full Text]

				B. Y. Ho, A. Stadnicka, P. L. Prather, A. R. Buckley, L. L. Current, Z. J. Bosnjak, and W.-M. Kwok Cannabinoid CB1 Receptor-Mediated Inhibition of Prolactin Release and Signaling Mechanisms in GH4C1 Cells Endocrinology, May 1, 2000; 141(5): 1675 - 1685. [Abstract] [Full Text] [PDF]

				P. L. Prather, N. A. Martin, C. S. Breivogel, and S. R. Childers Activation of Cannabinoid Receptors in Rat Brain by WIN 55212-2 Produces Coupling to Multiple G Protein alpha -Subunits with Different Potencies Mol. Pharmacol., May 1, 2000; 57(5): 1000 - 1010. [Abstract] [Full Text]

				L. Oliveira, A.C.M. Paiva, and G. Vriend A low resolution model for the interaction of G proteins with G protein-coupled receptors Protein Eng. Des. Sel., December 1, 1999; 12(12): 1087 - 1095. [Abstract] [Full Text] [PDF]

				M. Filizola, L. Laakkonen, and G. H. loew 3D modeling, ligand binding and activation studies of the cloned mouse {delta}, {micro} and {kappa} opioid receptors Protein Eng. Des. Sel., November 1, 1999; 12(11): 927 - 942. [Abstract] [Full Text] [PDF]

				P.-Y. Law and H. H. Loh Regulation of Opioid Receptor Activities J. Pharmacol. Exp. Ther., May 1, 1999; 289(2): 607 - 624. [Abstract] [Full Text]

				G. Griffin, P. J. Atkinson, V. M. Showalter, B. R. Martin, and M. E. Abood Evaluation of Cannabinoid Receptor Agonists and Antagonists Using the Guanosine-5'-O-(3-[35S]thio)-triphosphate Binding Assay in Rat Cerebellar Membranes J. Pharmacol. Exp. Ther., May 1, 1998; 285(2): 553 - 560. [Abstract] [Full Text]

				P. G. Szekeres and J. R. Traynor Delta Opioid Modulation of the Binding of Guanosine-5'-O-(3-[35S]thio)triphosphate to NG108-15 Cell Membranes: Characterization of Agonist and Inverse Agonist Effects J. Pharmacol. Exp. Ther., December 1, 1997; 283(3): 1276 - 1284. [Abstract] [Full Text]

				M. J. Clark, P. J. Emmerson, A. Mansour, H. Akil, J. H. Woods, P. S. Portoghese, A. E. Remmers, and F. Medzihradsky Opioid Efficacy in a C6 Glioma Cell Line Stably Expressing the Delta Opioid Receptor J. Pharmacol. Exp. Ther., November 1, 1997; 283(2): 501 - 510. [Abstract] [Full Text]

				S. Pepe, R.-P. Xiao, C. Hohl, R. Altschuld, and E. G. Lakatta `Cross Talk' Between Opioid Peptide and Adrenergic Receptor Signaling in Isolated Rat Heart Circulation, April 15, 1997; 95(8): 2122 - 2129. [Abstract] [Full Text]

				J. Nasman, C. C. Jansson, and K. E.O. Akerman The Second Intracellular Loop of the alpha 2-Adrenergic Receptors Determines Subtype-specific Coupling to cAMP Production J. Biol. Chem., April 11, 1997; 272(15): 9703 - 9708. [Abstract] [Full Text] [PDF]

				G. Martin, Z. Nie, and G. R. Siggins µ-Opioid Receptors Modulate NMDA Receptor-Mediated Responses in Nucleus Accumbens Neurons J. Neurosci., January 1, 1997; 17(1): 11 - 22. [Abstract] [Full Text] [PDF]

				S.-H. Chueh, S.-L. Song, and T.-Y. Liu Heterologous Desensitization of Opioid-stimulated Ca[IMAGE] Increase by Bradykinin or ATP in NG108-15 Cells J. Biol. Chem., July 14, 1995; 270(28): 16630 - 16637. [Abstract] [Full Text] [PDF]

				J.-G. Liu, M. B. Ruckle, and P. L. Prather Constitutively Active {micro}-Opioid Receptors Inhibit Adenylyl Cyclase Activity in Intact Cells and Activate G-proteins Differently than the Agonist [D-Ala2,N-MePhe4,Gly-ol5]Enkephalin J. Biol. Chem., October 5, 2001; 276(41): 37779 - 37786. [Abstract] [Full Text] [PDF]