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Pharmacological Analysis of Human D1 and D2 Dopamine Receptor Missense Variants

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Abstract

Drugs targeting dopamine receptors have been the focus of much research over the past 30 years, in large part because of their role in treating multiple pathological conditions including Parkinson’s disease, schizophrenia, Tourette’s syndrome, and hyperprolactinemia. Missense mutations in G protein-coupled receptors (GPCRs) can alter basal and/or ligand-induced signaling, which in turn can affect individuals’ susceptibility to disease and/or response to therapeutics. To date, five coding variants in the human D1 receptor (hD1R; T37P, T37R, R50S, S199A, and A229T) and three in the human D2 receptor (hD2R; P310S, S311C, and T351A) have been reported in the NCBI single nucleotide polymorphism database. We utilized site-directed mutagenesis to generate cDNAs encoding these receptor isoforms. After expression in either HEK293 or neuronal GT1 cells, basal and ligand-induced signaling of each of these receptors was determined and compared to wild type. In addition, we investigated expression levels of each recombinant receptor and the effect of inverse agonist administration. Our data demonstrate that naturally occurring amino acid substitutions in the hD1R can lead to alterations in expression levels as well as in basal and ligand-induced signaling. The potency and efficacy of dopamine, synthetic agonists (i.e., fenoldopam, SKF-38393, SKF-82958, and SCH23390), and inverse agonists [i.e., flupenthixol and (+)butaclamol] were reduced at selected hD1R variants. Furthermore, inverse agonist induced effects on expression levels were sensitive to selected amino acid substitutions. In contrast to the hD1R variants, hD2R polymorphisms did not affect ligand function or receptor expression. The observation that the hD1R mutations induce significant alterations in pharmacologic properties may have implications both for disease susceptibility and/or therapeutic response to dopaminergic ligands.

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Abbreviations

hD1R:

human dopamine D1 receptor

hD2R:

human dopamine D2 receptor

GPCR:

G protein-coupled receptor

TM:

transmembrane domain

HEK:

human embryonic kidney

SRE:

serum response element

CRE:

cyclic AMP response element

cAMP:

cyclic AMP

DA:

dopamine

DAR:

dopamine receptor

PD:

Parkinson’s disease

References

  • Al-Fulaij, M. A., Ren, Y., Beinborn, M., & Kopin, A. S. (2007). Identification of amino acid determinants of dopamine 2 receptor synthetic agonist function. Journal of Pharmacology and Experimental Therapeutics, 4, 4.

    Google Scholar 

  • Bach, T., Syversveen, T., Kvingedal, A. M., Krobert, K. A., Brattelid, T., Kaumann, A. J., et al. (2001). 5HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle. Naunyn Schmiedebergs Archives of Pharmacology, 363, 146–160.

    Article  CAS  Google Scholar 

  • Bai, M., Quinn, S., Trivedi, S., Kifor, O., Pearce, S. H., Pollak, M. R., et al. (1996). Expression and characterization of inactivating and activating mutations in the human Ca2+o-sensing receptor. Journal of Biological Chemistry, 271, 19537–19545.

    Article  PubMed  CAS  Google Scholar 

  • Beinborn, M., Lee, Y. M., McBride, E. W., Quinn, S. M., & Kopin, A. S. (1993). A single amino acid of the cholecystokinin-B/gastrin receptor determines specificity for non-peptide antagonists. Nature, 362, 348–350.

    Article  PubMed  CAS  Google Scholar 

  • Beinborn, M., Ren, Y., Blaker, M., Chen, C., & Kopin, A. S. (2004). Ligand function at constitutively active receptor mutants is affected by two distinct yet interacting mechanisms. Molecular Pharmacology, 65, 753–760.

    Article  PubMed  CAS  Google Scholar 

  • Birnbaumer, M., Gilbert, S., & Rosenthal, W. (1994). An extracellular congenital nephrogenic diabetes insipidus mutation of the vasopressin receptor reduces cell surface expression, affinity for ligand, and coupling to the Gs/adenylyl cyclase system. Molecular Endocrinology, 8, 886–894.

    Article  PubMed  CAS  Google Scholar 

  • Bond, R. A., & Ijzerman, A. P. (2006). Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends in Pharmacological Sciences, 27, 92–96.

    Article  PubMed  CAS  Google Scholar 

  • Bond, C., LaForge, K. S., Tian, M., Melia, D., Zhang, S., Borg, L., et al. (1998). Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proceedings of the National Academy of Sciences of the United States of America, 95, 9608–9613.

    Article  PubMed  CAS  Google Scholar 

  • Cai, G., Gurdal, H., Smith, C., Wang, H. Y., & Friedman, E. (1999). Inverse agonist properties of dopaminergic antagonists at the D(1A) dopamine receptor: uncoupling of the D(1A) dopamine receptor from G(s) protein. Molecular Pharmacology, 56, 989–996.

    PubMed  CAS  Google Scholar 

  • Claeysen, S., Joubert, L., Sebben, M., Bockaert, J., & Dumuis, A. (2003). A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL). Journal of Biological Chemistry, 278, 699–702.

    Article  PubMed  CAS  Google Scholar 

  • Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., & Bourne, H. R. (1993). Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature, 363, 274–276.

    Article  PubMed  CAS  Google Scholar 

  • Cravchik, A., Sibley, D. R., & Gejman, P. V. (1996). Functional analysis of the human D2 dopamine receptor missense variants. Journal of Biological Chemistry, 271, 26013–26017.

    Article  PubMed  CAS  Google Scholar 

  • Daeffler, L., & Landry, Y. (2000). Inverse agonism at heptahelical receptors: concept, experimental approach and therapeutic potential. Fundamental and Clinical Pharmacology, 14, 73–87.

    Article  PubMed  CAS  Google Scholar 

  • Emilien, G., Maloteaux, J. M., Geurts, M., Hoogenberg, K., & Cragg, S. (1999). Dopamine receptors–physiological understanding to therapeutic intervention potential. Pharmacology and Therapeutics, 84, 133–156.

    Article  PubMed  CAS  Google Scholar 

  • Feuerbach, D., Fehlmann, D., Nunn, C., Siehler, S., Langenegger, D., Bouhelal, R., et al. (2000). Cloning, expression and pharmacological characterisation of the mouse somatostatin sst(5) receptor. Neuropharmacology, 39, 1451–1462.

    Article  PubMed  CAS  Google Scholar 

  • Grandy, D. K., Marchionni, M. A., Makam, H., Stofko, R. E., Alfano, M., Frothingham, L., et al. (1989). Cloning of the cDNA and gene for a human D2 dopamine receptor. Proceedings of the National Academy of Sciences of the United States of America, 86, 9762–9766.

    Article  PubMed  CAS  Google Scholar 

  • Hawtin, S. R. (2006). Pharmacological chaperone activity of SR49059 to functionally recover misfolded mutations of the vasopressin V1a receptor. Journal of Biological Chemistry, 281, 14604–14614.

    Article  PubMed  CAS  Google Scholar 

  • Hearn, M. G., Ren, Y., McBride, E. W., Reveillaud, I., Beinborn, M., & Kopin, A. S. (2002). A Drosophila dopamine 2-like receptor: Molecular characterization and identification of multiple alternatively spliced variants. Proceedings of the National Academy of Sciences of the United States of America, 99, 14554–14559.

    Article  PubMed  CAS  Google Scholar 

  • Hu, J., Jiang, J., Costanzi, S., Thomas, C., Yang, W., Feyen, J. H., et al. (2006). A missense mutation in the seven-transmembrane domain of the human Ca2+receptor converts a negative allosteric modulator into a positive allosteric modulator. Journal of Biological Chemistry, 281, 21558–21565.

    Article  PubMed  CAS  Google Scholar 

  • Hyttel, J. (1983). SCH 23390 - the first selective dopamine D-1 antagonist. European Journal of Pharmacology, 91, 153–154.

    Article  PubMed  CAS  Google Scholar 

  • Insel, P. A., Tang, C. M., Hahntow, I., & Michel, M. C. (2006). Impact of GPCRs in clinical medicine: Monogenic diseases, genetic variants and drug targets. Biochimica et Biophysica Acta, 5, 5.

    Google Scholar 

  • Itokawa, M., Arinami, T., Futamura, N., Hamaguchi, H., & Toru, M. (1993). A structural polymorphism of human dopamine D2 receptor, D2(Ser311–>Cys). Biochemical and Biophysical Research Communications, 196, 1369–1375.

    Article  PubMed  CAS  Google Scholar 

  • Izenwasser, S., & Katz, J. L. (1993). Differential efficacies of dopamine D1 receptor agonists for stimulating adenylyl cyclase in squirrel monkey and rat. European Journal of Pharmacology, 246, 39–44.

    Article  PubMed  CAS  Google Scholar 

  • Kenakin, T. (2001). Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB Journal, 15, 598–611.

    Article  PubMed  CAS  Google Scholar 

  • Kopin, A. S., McBride, E. W., Chen, C., Freidinger, R. M., Chen, D., Zhao, C. M., et al. (2003). Identification of a series of CCK-2 receptor nonpeptide agonists: sensitivity to stereochemistry and a receptor point mutation. Proceedings of the National Academy of Sciences of the United States of America, 100, 5525–5530.

    Article  PubMed  CAS  Google Scholar 

  • Leineweber, K. (2004). Beta-adrenergic receptor polymorphism in human cardiovascular disease. Annals of Medicine, 36(Suppl 1), 64–69.

    Article  PubMed  CAS  Google Scholar 

  • Lin, C. W., Miller, T. R., Witte, D. G., Bianchi, B. R., Stashko, M., Manelli, A. M., et al. (1995). Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells. Molecular Pharmacology, 47, 131–139.

    PubMed  CAS  Google Scholar 

  • Maack, C., Cremers, B., Flesch, M., Hoper, A., Sudkamp, M., & Bohm, M. (2000). Different intrinsic activities of bucindolol, carvedilol and metoprolol in human failing myocardium. British Journal of Pharmacology, 130, 1131–1139.

    Article  PubMed  CAS  Google Scholar 

  • Milligan, G., & Bond, R. A. (1997). Inverse agonism and the regulation of receptor number. Trends in Pharmacological Sciences, 18, 468–474.

    PubMed  CAS  Google Scholar 

  • Missale, C., Nash, S. R., Robinson, S. W., Jaber, M., & Caron, M. G. (1998). Dopamine receptors: From structure to function. Physiological Reviews, 78, 189–225.

    PubMed  CAS  Google Scholar 

  • Oksche, A., & Rosenthal, W. (1998). The molecular basis of nephrogenic diabetes insipidus. Journal of Molecular Medicine, 76, 326–337.

    Article  PubMed  CAS  Google Scholar 

  • Peeters, R. P., van der Deure, W. M., & Visser, T. J. (2006). Genetic variation in thyroid hormone pathway genes; polymorphisms in the TSH receptor and the iodothyronine deiodinases. European Journal of Endocrinology, 155, 655–662.

    Article  PubMed  CAS  Google Scholar 

  • Ram, A., Cao, Q., Keck, P. E., Jr., Pope, H. G., Jr., Otani, K., Addonizio, G., et al. (1995). Structural change in dopamine D2 receptor gene in a patient with neuroleptic malignant syndrome. American Journal Medical Genetics, 60, 228–230.

    Article  CAS  Google Scholar 

  • Schulz, D. W., Staples, L., & Mailman, R. B. (1985). SCH23390 causes persistent antidopaminergic effects in vivo: evidence for longterm occupation of receptors. Life Sciences, 36, 1941–1948.

    Article  PubMed  CAS  Google Scholar 

  • Seifert, R., & Wenzel-Seifert, K. (2002). Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild type receptors. Naunyn Schmiedebergs Archives of Pharmacology, 366, 381–416.

    Article  CAS  Google Scholar 

  • Shinyama, H., Masuzaki, H., Fang, H., & Flier, J. S. (2003). Regulation of melanocortin-4 receptor signaling: agonist-mediated desensitization and internalization. Endocrinology, 144, 1301–1314.

    Article  PubMed  CAS  Google Scholar 

  • Tanaka, H., Moroi, K., Iwai, J., Takahashi, H., Ohnuma, N., Hori, S., et al. (1998). Novel mutations of the endothelin B receptor gene in patients with Hirschsprung’s disease and their characterization. Journal of Biological Chemistry, 273, 11378–11383.

    Article  PubMed  CAS  Google Scholar 

  • Tiberi, M., & Caron, M. G. (1994). High agonist-independent activity is a distinguishing feature of the dopamine D1B receptor subtype. Journal of Biological Chemistry, 269, 27925–27931.

    PubMed  CAS  Google Scholar 

  • Vaisse, C., Clement, K., Durand, E., Hercberg, S., Guy-Grand, B., & Froguel, P. (2000). Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. Journal of Clinical Investigation, 106, 253–262.

    Article  PubMed  CAS  Google Scholar 

  • Vallone, D., Picetti, R., & Borrelli, E. (2000). Structure and function of dopamine receptors. Neuroscience and Biobehavioral Reviews, 24, 125–132.

    Article  PubMed  CAS  Google Scholar 

  • Weiner, R. I., Wetsel, W., Goldsmith, P., Martinez de la Escalera, G., Windle, J., Padula, C., et al. (1992). Gonadotropin-releasing hormone neuronal cell lines. Frontiers in Neuroendocrinology, 13, 95–119.

    PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Dr. Grandy and Dr. Bunzow for kindly providing us with the human D2 receptor cDNA. We would also like to thank Dr. Weiner for providing us with the GT1 cells. This work was supported by the National Institute of Health grants, R01-DA020415 and R01-DK072497, and Digestive Disease Research Center grant, P30-DK34928.

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Authors and Affiliations

  1. Program in Genetics, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA, 02111, USA

    Munya A. Al-Fulaij & Alan S. Kopin

  2. Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts-New England Medical Center, 750 Washington Street, Box 7703, Boston, MA, 02111, USA

    Yong Ren, Martin Beinborn & Alan S. Kopin

Authors
  1. Munya A. Al-Fulaij
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  2. Yong Ren
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  4. Alan S. Kopin
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Correspondence to Alan S. Kopin.

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Al-Fulaij, M.A., Ren, Y., Beinborn, M. et al. Pharmacological Analysis of Human D1 and D2 Dopamine Receptor Missense Variants. J Mol Neurosci 34, 211–223 (2008). https://doi.org/10.1007/s12031-007-9030-x

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  • DOI: https://doi.org/10.1007/s12031-007-9030-x

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