|
Direct Supervisor: Federico Mayor, jr., MD
Location: Departamento de Biología Molecular and Centro de Biología Molecular “Severo Ochoa”, Madrid.
Descriptive title of research activity: Role of G protein-coupled receptor kinases (GRKs) in cell migration and adhesion
Overall goals: The G protein -coupled receptor kinases (GRKs) are key regulators of multiple G protein-coupled receptors (GPCR). Our main goal is to study in a deeper level the functional role of GRK2 in cell migration and adhesion, and to investigate the functional consequences of altering its levels, or its specific interaction with defined proteins involved in cell migration.
Rationale and significance: GPCR mediate the actions of a variety of messengers that are key regulators of the immune system such as chemokines, prostaglandins, or cathecholamines. G protein-coupled receptor kinases (GRKs) are serine-threonine kinases that phosphorylate agonist-occupied GPCR, thus promoting the binding of the cytosolic -arrestins to the phosphorylated receptor. This results in receptor uncoupling from G proteins (desensitization) and also triggers GPCR endocytosis. In addition to this role, recent evidence suggests that both GRKs and arrestins have important scaffold and signal-propagating functions. Thus, GRKs may play a direct role in GPCR signaling, by phosphorylating non-GPCR substrates and by recruiting other cellular proteins. In this regard, GRKs have been recently shown to interact with several proteins that play important roles in cell migration ,as G protein subunits (Gq and Gbetagamma), PI3K and GIT, a multidomain protein with Arf-GAP activity able to interact with Rac GTPase modulators (Cool/Pix), paxillin and MEK1.The significance of these functional interactions is only partially known, but they allow GRK2 to control the activity, location, stability or effector interactions of its interacting proteins in a manner independent of phosphorylation.
Description of work and methodology: Data from our lab established that the interaction between GIT and GRK2 is modulated upon phosphorylation by different kinases and that changes in the levels of GRK2 alter the cytoskeletal morphology in epithelial cells and promote changes in fibronectin-dependent cell migration in a manner independent of GRK2 kinase activity. In this context, we plan to: a) Investigate the participation of GRK2 in integrin-mediated cell migration and motility analyzing the effects of altering the levels, activity or interaction of GRK2 with GIT; b) Explore the effect of the activation by integrins on the phosphorylation of GRK2, its association with GIT and on the GRK2 cellular dynamics, as well as GRK2 recruitment to integrin signaling complexes and the mechanisms thereby implicated; c) Explore in detail the alterations in cytoskeletal morphology and the related signaling pathways promoted by changes in the expression of GRK2; d) Characterize the domains implicated in the GRK2/GIT interaction, the cellular stimuli (integrins, chemokine receptors, receptors coupled to Gq) that modulate its dynamic association/dissociation and its subcellular localization and the cellular consequences of impairing the GIT-GRK2 interaction; e) Explore the participation of GRK2 in the transmodulation of integrin receptors by chemokine receptors and viceversa.
Methodology: For addressing the issues mentioned above, we will use a combination of molecular and cell biology techniques, in collaboration with other groups of the MAIN network. Experimental approaches include: adhesion assays in cell lines stably over-expressing GRK2 or different kinase mutants, spreading assays by light microscopy or immunofluorescence microscopy, FACS analysis, immunoblotting, co-immunoprecipitation and signal transduction assays, confocal microscopy and dynamic localization of GFP constructs of the proteins of interest, cell migration assays, etc. knockouts or knockdown models will be used with altered GRK2 and/or GIT levels (by either overexpression or siRNA-mediated knock-down or by using cells obtained from available heterozygous GRK2 knockout mice).
|
