The cytoplasmic tail of the D1A receptor subtype: identification of specific domains controlling dopamine cellular responsiveness.
In this study the rat D1A receptor (wild-type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor-mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Delta425, Delta379, Delta351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys(351) and Asp(425) may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Delta351 is significantly increased in comparison with WT, Delta425 and Delta379. Furthermore, we demonstrate that the extent of dopamine-mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Delta351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys(351) may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Delta351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.
Chaar
Z Y
ZY
Ottawa Health Research Institute, and Departments of Medicine/Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Jackson
A
A
Tiberi
M
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D004298:Dopamine / Q000378:metabolism*; D018491:Dopamine Agonists / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018491:Dopamine Agonists; D011956:Receptors, Cell Surface; D017447:Receptors, Dopamine D1; C098593:dopamine D1A receptor; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004298:Dopamine