title: The C. elegans VIG-1 and FRM-1 modulate carbachol-stimulated ERK1/2 activation in chinese hamster ovary cells expressing the muscarinic acetylcholine receptor GAR-3.
abstract: Many neurotransmitter receptors are known to interact with a variety of intracellular proteins that modulate signaling processes. In an effort to understand the molecular mechanism by which acetylcholine (ACh) signaling is modulated, we searched for proteins that interact with GAR-3, the Caenorhabditis elegans homolog of muscarinic ACh receptors. We isolated two proteins, VIG-1 and FRM-1, in a yeast two-hybrid screen of a C. elegans cDNA library using the third intracellular (i3) loop of GAR-3 as bait. To test whether these proteins regulate ACh signaling, we utilized Chinese hamster ovary (CHO) cells stably expressing GAR-3 (GAR-3/CHO cells). Previously we have shown that the cholinergic agonist carbachol stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in an atropine-sensitive manner in this cell line. When VIG-1 was transiently expressed in GAR-3/CHO cells, carbachol-stimulated ERK1/2 activation was substantially reduced. In contrast, transient expression of FRM-1 significantly enhanced carbachol-stimulated ERK1/2 activation. Neither VIG-1 nor FRM-1 expression appeared to alter the affinity between GAR-3 and carbachol. In support of this notion, expression of these proteins did not affect GAR-3-mediated phospholipase C activation. To verify the modulation of ERK1/2 activity by VIG-1 and FRM-1, we used an i3 loop deletion mutant of GAR-3 (termed GAR-3Δi3). Carbachol treatment evoked robust ERK1/2 activation in CHO cells stably expressing the deletion mutant (GAR-3Δi3/CHO cells). However, transient expression of either VIG-1 or FRM-1 had little effect on carbachol-stimulated ERK1/2 activation in GAR-3Δi3/CHO cells. Taken together, these results indicate that VIG-1 and FRM-1 regulate GAR-3-mediated ERK1/2 activation by interacting with the i3 loop of GAR-3.
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju, 361-763, South Korea.
authors.forename: Youngmi
authors.initials: Y
authors.lastname: Shin
authors.forename: Nam Jeong
authors.initials: NJ
authors.lastname: Cho
mesh_terms: D000818:Animals; D016466:CHO Cells; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000819:agonists / Q000096:biosynthesis* / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000096:biosynthesis*; D012261:Ribonucleoproteins / Q000502:physiology*
chemical_list: D029742:Caenorhabditis elegans Proteins; D018679:Cholinergic Agonists; C477876:GAR-3 protein, C elegans; D011976:Receptors, Muscarinic; D012261:Ribonucleoproteins; C539742:VIG-1 protein, C elegans; D002217:Carbachol