title: Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling.
abstract: Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP-Galpha and Gbetagamma subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP-Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the beta subunit of heterotrimeric G proteins in vitro, and with the betagamma subunit complex in vivo [6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gbeta1gamma2, and that this association in mammalian cells is significantly enhanced when active p21(Ras) is present or when cRaf1 is otherwise targeted to the membrane. Association with Gbeta1gamma2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gbetagamma-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cbeta (PLCbeta) by sequestration of Gbetagamma subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gbetagamma, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21(Ras).
authors.affiliation: Institut für medizinische Strahlenkunde und Zellforschung (MSZ) der Universität Würzburg Versbacherstrasse 5, D-97078, Würzburg, Germany.
authors.forename: J R
authors.initials: JR
authors.lastname: Slupsky
authors.forename: U
authors.initials: U
authors.lastname: Quitterer
authors.forename: C K
authors.initials: CK
authors.lastname: Weber
authors.forename: P
authors.initials: P
authors.lastname: Gierschik
authors.forename: M J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: U R
authors.initials: UR
authors.lastname: Rapp
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D020935:MAP Kinase Signaling System / Q000502:physiology*; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection
chemical_list: D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf; D020962:Heterotrimeric GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)