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Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling
J R Slupsky1, U Quitterer, C K Weber
1Institut für medizinische Strahlenkunde und Zellforschung (MSZ) der Universität Würzburg Versbacherstrasse 5, D-97078, Würzburg, Germany.
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).
Insights
Cellular signaling involves protein kinase cRaf1 interacting with Gbetagamma subunits. Membrane-localized cRaf1 inhibits G-protein-coupled receptor signaling by sequestering Gbetagamma, regulating cellular responses.
Area of Science:
- Cellular signaling pathways
- G-protein coupled receptor (GPCR) signaling
- Mitogenic signaling cascades
Background:
- Seven transmembrane domain receptors activate heterotrimeric G proteins, initiating cellular responses.
- Mitogen-activated protein (MAP) kinase cascade, initiated by cRaf1, is crucial for cell growth and differentiation.
- The interaction between cRaf1 and G-protein subunits was previously reported but its functional role remained unclear.
Purpose of the Study:
- To investigate the association between cRaf1 and Gbetagamma subunits in mammalian cells.
- To determine the functional consequences of cRaf1-Gbetagamma interaction on cellular signaling.
- To elucidate the role of cRaf1 in regulating G-protein-coupled receptor (GPCR) mediated signaling.
Main Methods:
- Co-immunoprecipitation assays to study protein-protein interactions.
- Cellular localization studies to assess membrane recruitment of cRaf1.
- Functional assays to measure phospholipase Cbeta (PLCbeta) activation and Gbetagamma-mediated signaling.
Main Results:
- cRaf1 directly associates with Gbeta1gamma2 subunits.
- This association is enhanced by the presence of active Ras or membrane targeting of cRaf1.
- Membrane-localized cRaf1 inhibits GPCR-stimulated PLCbeta activation by sequestering Gbetagamma subunits.
- cRaf1 binding to Gbetagamma does not affect cRaf1 kinase activity but modulates Gbetagamma signaling.
Conclusions:
- cRaf1 acts as a regulator of Gbetagamma signaling, particularly in GPCR pathways activating the MAP kinase cascade.
- Membrane recruitment of cRaf1 plays a key role in modulating Gbetagamma-mediated cellular responses.
- These findings highlight a novel mechanism by which cRaf1 influences signal transduction downstream of GPCRs.