Allosteric communication between protomers of dopamine Class A GPCR dimers modulates activation

A major obstacle to understanding the functional importance of dimerization between Class A G protein-coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control and illustrate it for the human dopamine D2 receptor. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer. Inverse agonist binding to the second protomer enhances signaling, whereas agonist binding to the second protomer blunts signaling. Ligand-independent constitutive activation of the second protomer also inhibits signaling. Thus, GPCR dimer function can be modulated by the activity state of the second protomer, which for a heterodimer may be altered in pathological states. Our novel methodology also makes possible the characterization of signaling from a defined heterodimer unit.

[1]  J. Ballesteros,et al.  [19] Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors , 1995 .

[2]  J. Pin,et al.  G Protein Activation by the Leukotriene B4 Receptor Dimer , 2008, Journal of Biological Chemistry.

[3]  H. Hamm,et al.  The 2.0 Å crystal structure of a heterotrimeric G protein , 1996, Nature.

[4]  P. Molinari,et al.  Promiscuous Coupling at Receptor-Gα Fusion Proteins , 2003, The Journal of Biological Chemistry.

[5]  A M J J Bonvin,et al.  Data‐driven docking: HADDOCK's adventures in CAPRI , 2005, Proteins.

[6]  S. Costagliola,et al.  Structure-function relationships of two loss-of-function mutations of the thyrotropin receptor gene. , 1999, Thyroid : official journal of the American Thyroid Association.

[7]  Marta Filizola,et al.  Modeling activated states of GPCRs: the rhodopsin template , 2007, J. Comput. Aided Mol. Des..

[8]  L. Prézeau,et al.  Closed state of both binding domains of homodimeric mGlu receptors is required for full activity , 2004, Nature Structural &Molecular Biology.

[9]  Ann Allergy,et al.  O R I G I N a L a R T I C L E S , 2022 .

[10]  A. Sali,et al.  Comparative protein structure modeling of genes and genomes. , 2000, Annual review of biophysics and biomolecular structure.

[11]  Marta Filizola,et al.  Dopamine D2 receptors form higher order oligomers at physiological expression levels , 2008, The EMBO journal.

[12]  H G Khorana,et al.  Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show position-specific effects in transducin activation. , 1996, Biochemistry.

[13]  G. Milligan,et al.  Functional Complementation and the Analysis of Opioid Receptor Homodimerization , 2005, Molecular Pharmacology.

[14]  Graeme Milligan,et al.  Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins* , 2003, Journal of Biological Chemistry.

[15]  G. Marshall,et al.  Rhodopsin-transducin interface: studies with conformationally constrained peptides. , 2001, Biophysical journal.

[16]  Graeme Milligan,et al.  Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric beta2-adrenoceptor. , 2007, Cellular signalling.

[17]  M. Moussaif,et al.  Rhodopsin Determinants for Transducin Activation , 2003, Journal of Biological Chemistry.

[18]  G. Milligan,et al.  CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2–DOP receptor heterodimer , 2008, The Biochemical journal.

[19]  A. Bax,et al.  Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings. , 2002, Journal of molecular biology.

[20]  Leonardo Pardo,et al.  An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors , 2005, Journal of Biological Chemistry.

[21]  L. Prézeau,et al.  Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. , 2003, Pharmacology & therapeutics.

[22]  J. Banères,et al.  Cooperative Conformational Changes in a G-protein-coupled Receptor Dimer, the Leukotriene B4 Receptor BLT1* , 2004, Journal of Biological Chemistry.

[23]  Marta Filizola,et al.  Crosstalk in G protein-coupled receptors: changes at the transmembrane homodimer interface determine activation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Rees,et al.  A cysteine-3 to serine mutation of the G-protein Gi1 alpha abrogates functional activation by the alpha 2A-adrenoceptor but not interactions with the beta gamma complex. , 1997, Biochemistry.

[25]  H Weinstein,et al.  Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the μ opioid receptor , 1999, FEBS letters.

[26]  J. Ballesteros,et al.  The first transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle. , 2000, Biochemistry.

[27]  M. A. Downs,et al.  Rhodopsin-interacting surface of the transducin γ subunit , 2006 .

[28]  C. Strader,et al.  Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity. , 1987, The Journal of biological chemistry.

[29]  J. Pin,et al.  Asymmetric conformational changes in a GPCR dimer controlled by G‐proteins , 2006, The EMBO journal.

[30]  L. Prézeau,et al.  Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR , 2005, The EMBO journal.

[31]  Holger Gohlke,et al.  The Amber biomolecular simulation programs , 2005, J. Comput. Chem..

[32]  B. O'dowd,et al.  Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. , 2000, The Journal of biological chemistry.

[33]  H Weinstein,et al.  The fourth transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle. , 2000, Biochemistry.

[34]  R Seifert,et al.  Restricting the mobility of Gs alpha: impact on receptor and effector coupling. , 1999, Biochemistry.

[35]  E. Weiss,et al.  The Effect of Carboxyl-terminal Mutagenesis of G on Rhodopsin and Guanine Nucleotide Binding (*) , 1995, The Journal of Biological Chemistry.

[36]  D. Farrens,et al.  Rhodopsin Activation Exposes a Key Hydrophobic Binding Site for the Transducin α-Subunit C Terminus* , 2004, Journal of Biological Chemistry.

[37]  K. Neve,et al.  Modeling and mutational analysis of a putative sodium-binding pocket on the dopamine D2 receptor. , 2001, Molecular pharmacology.

[38]  B. Conklin,et al.  Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. , 1993, Nature.

[39]  R Marsault,et al.  Transfected Aequorin in the Measurement of Cytosolic Ca2+ Concentration ([Ca2+]c) , 1995, The Journal of Biological Chemistry.

[40]  T. Sakmar,et al.  The Amino Terminus of the Fourth Cytoplasmic Loop of Rhodopsin Modulates Rhodopsin-Transducin Interaction* , 2000, The Journal of Biological Chemistry.

[41]  Tullio Pozzan,et al.  Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin , 1992, Nature.

[42]  H. Bourne,et al.  A mutation that prevents GTP-dependent activation of the α chain of Gs , 1988, Nature.

[43]  M. Parmentier,et al.  Allosteric Modulation of Binding Properties between Units of Chemokine Receptor Homo- and Hetero-Oligomers , 2006, Molecular Pharmacology.

[44]  Richard N. Zare,et al.  A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein , 2007, Proceedings of the National Academy of Sciences.

[45]  J. Ballesteros,et al.  Dopamine D4/D2 receptor selectivity is determined by A divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments. , 1999, Molecular pharmacology.

[46]  B. Kobilka,et al.  GPCR-Galpha fusion proteins: molecular analysis of receptor-G-protein coupling. , 1999, Trends in pharmacological sciences.

[47]  G Vassart,et al.  Extracellular Cysteines of CCR5 Are Required for Chemokine Binding, but Dispensable for HIV-1 Coreceptor Activity* , 1999, The Journal of Biological Chemistry.

[48]  B. O'dowd,et al.  Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal* , 2004, Journal of Biological Chemistry.

[49]  Restricting mobility of Gsalpha relative to the beta2-adrenoceptor enhances adenylate cyclase activity by reducing Gsalpha GTPase activity. , 1998, The Biochemical journal.

[50]  M. Parmentier,et al.  Allosteric properties of G protein-coupled receptor oligomers. , 2007, Pharmacology & therapeutics.

[51]  W. Sadee,et al.  Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling. , 1993, The Journal of biological chemistry.

[52]  H. Khorana,et al.  Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Rolf Boelens,et al.  Information-driven protein–DNA docking using HADDOCK: it is a matter of flexibility , 2006, Nucleic acids research.

[54]  Francine Acher,et al.  Asymmetric Functioning of Dimeric Metabotropic Glutamate Receptors Disclosed by Positive Allosteric Modulators* , 2005, Journal of Biological Chemistry.

[55]  J. Javitch,et al.  Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. , 1995, Biochemistry.

[56]  N. Artemyev,et al.  Rhodopsin Determinants for Transducin Activation: A Gain-of-Function Approach , 2003 .

[57]  B. Kobilka,et al.  GPCR–Gα fusion proteins: molecular analysis of receptor–G-protein coupling , 1999 .

[58]  S. Karnik,et al.  Transducin-α C-terminal Peptide Binding Site Consists of C-D and E-F Loops of Rhodopsin* , 1997, The Journal of Biological Chemistry.

[59]  B. Conklin,et al.  Substitution of three amino acids switches receptor specificity of Gqα to that of Giα , 1993, Nature.

[60]  Olivier Lichtarge,et al.  Receptor and βγ Binding Sites in the α Subunit of the Retinal G Protein Transducin , 1997, Science.

[61]  H. Khorana,et al.  Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[62]  Lei Shi,et al.  The Fourth Transmembrane Segment Forms the Interface of the Dopamine D2 Receptor Homodimer* , 2003, The Journal of Biological Chemistry.

[63]  J. Ballesteros,et al.  Activation of the β2-Adrenergic Receptor Involves Disruption of an Ionic Lock between the Cytoplasmic Ends of Transmembrane Segments 3 and 6* , 2001, The Journal of Biological Chemistry.

[64]  O. Lichtarge,et al.  Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin. , 1997, Science.

[65]  J. Pin,et al.  Activation of a Dimeric Metabotropic Glutamate Receptor by Intersubunit Rearrangement* , 2007, Journal of Biological Chemistry.

[66]  K. Lorenz,et al.  Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling. , 2008, Nature chemical biology.

[67]  Michel Bouvier,et al.  International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the Recognition and Nomenclature of G Protein-Coupled Receptor Heteromultimers , 2007, Pharmacological Reviews.

[68]  J. Javitch,et al.  Mechanisms of inverse agonism of antipsychotic drugs at the D2 dopamine receptor: use of a mutant D2 dopamine receptor that adopts the activated conformation , 2001, Journal of neurochemistry.

[69]  P. Henklein,et al.  Mutation of the Fourth Cytoplasmic Loop of Rhodopsin Affects Binding of Transducin and Peptides Derived from the Carboxyl-terminal Sequences of Transducin α and γ Subunits* , 2000, The Journal of Biological Chemistry.

[70]  H. Khorana,et al.  Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[71]  Oliver P. Ernst,et al.  Crystal structure of opsin in its G-protein-interacting conformation , 2008, Nature.

[72]  D. Oprian,et al.  Transducin Activation by Nanoscale Lipid Bilayers Containing One and Two Rhodopsins* , 2007, Journal of Biological Chemistry.

[73]  P. Conn,et al.  Cis- and trans-activation of hormone receptors: the LH receptor. , 2002, Molecular endocrinology.

[74]  Arthur Christopoulos,et al.  Functional Selectivity and Classical Concepts of Quantitative Pharmacology , 2007, Journal of Pharmacology and Experimental Therapeutics.

[75]  R. Shigemoto,et al.  GABAB-receptor subtypes assemble into functional heteromeric complexes , 1998, Nature.