Modeling and mutational analysis of a putative sodium-binding pocket on the dopamine D2 receptor.

A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.

[1]  K. Jarnagin,et al.  Mutations in the B2 Bradykinin Receptor Reveal a Different Pattern of Contacts for Peptidic Agonists and Peptidic Antagonists* , 1996, Journal of Biological Chemistry.

[2]  Tom Alber,et al.  Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme , 1988, Nature.

[3]  J. Wess,et al.  Hydrophilic side chains in the third and seventh transmembrane helical domains of human A2A adenosine receptors are required for ligand recognition. , 1996, Molecular pharmacology.

[4]  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 .

[5]  Marvin C. Gershengorn,et al.  Interactions between Conserved Residues in Transmembrane Helices 1, 2, and 7 of the Thyrotropin-releasing Hormone Receptor* , 1997, The Journal of Biological Chemistry.

[6]  K. Neve,et al.  Contributions of Conserved Serine Residues to the Interactions of Ligands with Dopamine D2 Receptors , 1992, Journal of Neurochemistry.

[7]  K. Neve,et al.  Regulation of dopamine D2 receptors by sodium and pH. , 1991, Molecular pharmacology.

[8]  A. Scheer,et al.  Constitutively active mutants of the alpha 1B‐adrenergic receptor: role of highly conserved polar amino acids in receptor activation. , 1996, The EMBO journal.

[9]  M. Whitlow,et al.  An empirical examination of potential energy minimization using the well-determined structure of the protein crambin , 1986 .

[10]  X. P. Liu,et al.  The active state of the AT1 angiotensin receptor is generated by angiotensin II induction. , 1996, Biochemistry.

[11]  S. Snyder,et al.  Opiate Receptor Binding of Agonists and Antagonists Affected Differentially by Sodium , 1974 .

[12]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2000, Science.

[13]  R. Henderson,et al.  Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. , 1990, Journal of molecular biology.

[14]  L. Limbird,et al.  Sodium ion modulates agonist and antagonist interactions with the human platelet alpha 2-adrenergic receptor in membrane and solubilized preparations. , 1982, Molecular pharmacology.

[15]  P. Chanda,et al.  Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor. , 1993, Molecular pharmacology.

[16]  Y. Cheng,et al.  Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. , 1973, Biochemical pharmacology.

[17]  C. A. Guyer,et al.  An aspartate conserved among G-protein receptors confers allosteric regulation of alpha 2-adrenergic receptors by sodium. , 1990, The Journal of biological chemistry.

[18]  A. IJzerman,et al.  Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding. , 1996, Molecular pharmacology.

[19]  O. Lichtarge,et al.  C5a Receptor Activation , 1999, The Journal of Biological Chemistry.

[20]  Mark Froimowitz,et al.  Homology modeling of the dopamine D2 receptor and its testing by docking of agonists and tricyclic antagonists. , 1994, Journal of medicinal chemistry.

[21]  K. Neve,et al.  Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenylyl cyclase. , 1991, Molecular pharmacology.

[22]  J. Venter,et al.  Site-directed mutagenesis and continuous expression of human beta-adrenergic receptors. Identification of a conserved aspartate residue involved in agonist binding and receptor activation. , 1988, The Journal of biological chemistry.

[23]  H. Motulsky,et al.  Influence of sodium on the alpha 2-adrenergic receptor system of human platelets. Role for intraplatelet sodium in receptor binding. , 1983, The Journal of biological chemistry.

[24]  K. Neve,et al.  Sensitization of endogenous and recombinant adenylate cyclase by activation of D2 dopamine receptors. , 1996, Molecular pharmacology.

[25]  T. Shimizu,et al.  High and low affinity mutants of platelet-activating factor receptor. , 1997, Advances in experimental medicine and biology.

[26]  C. Strader,et al.  Identification of two serine residues involved in agonist activation of the beta-adrenergic receptor. , 1989, The Journal of biological chemistry.

[27]  Jonathan A Javitch,et al.  Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method , 1995, Neuron.

[28]  J. Ballesteros,et al.  Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice. , 1996, Biochemistry.

[29]  J. Vincent,et al.  Pivotal role of an aspartate residue in sodium sensitivity and coupling to G proteins of neurotensin receptors. , 1999, Molecular pharmacology.

[30]  H. Khorana,et al.  Requirement of Rigid-Body Motion of Transmembrane Helices for Light Activation of Rhodopsin , 1996, Science.

[31]  P. Devoto,et al.  Sodium-dependent interaction of benzamides with dopamine receptors , 1980, Brain Research.

[32]  W. Greenlee,et al.  Dual agonistic and antagonistic property of nonpeptide angiotensin AT1 ligands: susceptibility to receptor mutations. , 1997, Molecular pharmacology.

[33]  J. Venter,et al.  Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: conserved aspartic acids play important roles in receptor function. , 1989, Molecular pharmacology.

[34]  A. IJzerman,et al.  TinyGRAP database: a bioinformatics tool to mine G-protein-coupled receptor mutant data. , 1999, Trends in pharmacological sciences.

[35]  J. Nunnari,et al.  Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange. , 1987, The Journal of biological chemistry.

[36]  K. Jacobson,et al.  A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor. , 1997, Molecular pharmacology.

[37]  L. Limbird.  Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms , 1988, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[38]  Pierre Corvol,et al.  Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling , 1996, The Journal of Biological Chemistry.

[39]  E C Hulme,et al.  The Functional Topography of Transmembrane Domain 3 of the M1 Muscarinic Acetylcholine Receptor, Revealed by Scanning Mutagenesis* , 1999, The Journal of Biological Chemistry.

[40]  H. Khorana,et al.  Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state. , 1997, Proceedings of the National Academy of Sciences of the United States of America.