title: SH3 binding domains in the dopamine D4 receptor.
abstract: The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.
authors.affiliation: Department of Pharmacology, Institute of Medical Science, University of Toronto, Ontario, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Oldenhof
authors.forename: R
authors.initials: R
authors.lastname: Vickery
authors.forename: M
authors.initials: M
authors.lastname: Anafi
authors.forename: J
authors.initials: J
authors.lastname: Oak
authors.forename: A
authors.initials: A
authors.lastname: Ray
authors.forename: O
authors.initials: O
authors.lastname: Schoots
authors.forename: T
authors.initials: T
authors.lastname: Pawson
authors.forename: M
authors.initials: M
authors.lastname: von Zastrow
authors.forename: H H
authors.initials: HH
authors.lastname: Van Tol
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050638:Receptors, Dopamine D4; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D018909:src Homology Domains / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C493599:DRD4 protein, human; C493600:Drd4 protein, mouse; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C078332:Nck protein; D015513:Oncogene Proteins; D010446:Peptide Fragments; D011506:Proteins; D011956:Receptors, Cell Surface; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D050638:Receptors, Dopamine D4; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins