Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution.
Alternatively spliced variants of the D2 dopamine receptor have distinct neuronal function and localization. The long isoform (D2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop-a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Galphai/o protein coupling specificities. However, the exact role of the alternatively spliced insert region in D2 dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D2L mutant is indistinguishable from the wild type D2L receptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D2 receptor function.
Kendall
Ryan T
RT
University of Tennessee Health Science Center, College of Medicine, Department of Molecular Sciences, 858 Madison Avenue, G01, Memphis, TN 38163, USA.
Senogles
Susan E
SE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology*; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015259:Dopamine Agents / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry / Q000502:physiology; D006801:Humans; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008958:Models, Molecular; D012333:RNA, Messenger / Q000096:biosynthesis; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000502:physiology; D013134:Spiperone / Q000493:pharmacokinetics; D013997:Time Factors; D014162:Transfection / Q000379:methods
D015259:Dopamine Agents; D018492:Dopamine Antagonists; D000939:Epitopes; D012333:RNA, Messenger; D017448:Receptors, Dopamine D2; C113776:dopamine D2L receptor; D005576:Colforsin; D013134:Spiperone; D000242:Cyclic AMP; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases