title: The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids.
abstract: Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca(2+)-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent K(d, PIPx) of 2-5 nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.
authors.affiliation: Department of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217, USA.
authors.forename: Tatyana A
authors.initials: TA
authors.lastname: Lyakhova
authors.affiliation: Department of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217, USA. Electronic address: jefferson.knight@ucdenver.edu.
authors.forename: Jefferson D
authors.initials: JD
authors.lastname: Knight
mesh_terms: D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism*; D008958:Models, Molecular; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007328:Insulin; D008563:Membrane Lipids; D018129:Phosphatidylinositol Phosphates; C512779:SYTL4 protein, human; D033921:Vesicular Transport Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D010833:Phytic Acid
keywords: Inositol polyphosphate signaling; Insulin secretion; Phosphatidylinositol-(4,5)-bisphosphate; Protein–lipid interaction; Secretory granule docking; Slp4