title: Functional characterization of rare variants in human dopamine receptor D4 gene by genotype-phenotype correlations.
abstract: Next generation sequencing technologies have facilitated a notable shift from common disease common variant hypothesis to common disease rare variant, as also witnessed in recent literature on schizophrenia. Dopamine receptor D4 (DRD4), a G-protein-coupled receptor is associated with psychiatric disorders and has high affinity for atypical antipsychotic clozapine. We investigated the functional role of rare genetic variants in DRD4 which may have implications for translational medicine. CHO-K1 cells independently expressing four rare non-synonymous variants of DRD4 namely R237L, A281P, S284G located in the third cytosolic loop and V194G, located in the fifth transmembrane domain were generated. Their genotype-phenotype correlations were evaluated using [³H]spiperone binding, G-protein activation and molecular dynamics-simulation studies. A281P and S284G were functionally similar to wildtype (WT). With R237L, potency of dopamine and quinpirole reduced ∼sixfold and threefold respectively compared to WT; [³H]spiperone binding studies showed a reduction in total number of binding sites (∼40%) but not binding affinity, in silico docking studies revealed that binding of both dopamine and spiperone to R237L was structurally similar to WT. Of note, V194G variant failed to inhibit forskolin-stimulated adenylate cyclase activity and phosphorylate extracellular signal-regulated kinase; showed significant reduction in binding affinity (K(d)=2.16 nM) and total number of binding sites (∼66%) compared to WT in [³H]spiperone binding studies; and ligand docking studies showed that binding of dopamine and spiperone is superficial due to probable structural alteration. Transmembrane variant V194G in DRD4.4 results in functional alteration warranting continuing functional analysis of rare variants.
authors.affiliation: Department of Genetics, University of Delhi South Campus, New Delhi, India.
authors.forename: K A
authors.initials: KA
authors.lastname: Michealraj
authors.affiliation: Bioinformatics Infrastructure Facility, Sri Venkateswara College, New Delhi, India.
authors.forename: N
authors.initials: N
authors.lastname: Jatana
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.
authors.lastname: Jafurulla
authors.affiliation: Bioinformatics Infrastructure Facility, Sri Venkateswara College, New Delhi, India.
authors.forename: L
authors.initials: L
authors.lastname: Narayanan
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.
authors.forename: A
authors.initials: A
authors.lastname: Chattopadhyay
authors.affiliation: Department of Genetics, University of Delhi South Campus, New Delhi, India. Electronic address: thelmabk@gmail.com.
authors.forename: B K
authors.initials: BK
authors.lastname: Thelma
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D056726:Genetic Association Studies; D006801:Humans; D056004:Molecular Dynamics Simulation; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D017433:Protein Structure, Secondary; D050638:Receptors, Dopamine D4 / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: C493599:DRD4 protein, human; D050638:Receptors, Dopamine D4; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; D004298:Dopamine
keywords: G-protein-coupled receptors; dopamine receptor D4; genotype–phenotype correlations; ligand docking; rare variants