The functional role of Dynamin 2 and GRB2 in CCR5 mediated chemotaxis

Richard Jacques, Anja Mueller. University of East Anglia, Department of Pharmacy, Norwich Research Park, Norwich, NR4 7TJ, UK

The HIV viral entry co-receptor CCR5 functions physiologically as a typical CC motif chemokine receptor. Activation of CCR5 leads to cytosolic signal transduction which results in a variety of cellular responses such as intracellular calcium release, cytoskeletal rearrangement and chemotaxis.1 Attenuation of CCR5 signalling occurs through receptor phosphorylation and arrestin mediated desensitisation. Recycling or degradation then occurs by internalisation via caveolae or clathrin coated pits in a dynamin 2 (dyn2) dependent fashion.2, 3 The cytosolic events triggered by CCR5 activation however remain to be characterised. A better understanding of intracellular signalling events may be useful in understanding HIV infection, chemokine mediated inflammatory responses or GPCR signalling more generally.

Our aim was to investigate the signalling pathways involved in CCR5 mediated calcium release and chemotaxis, using calcium release and chemotaxis assays, actin polymerisation and internalisation visualisation in THP-1 (endogenous CCR5 expression) CHO.CCR5 and HeLa.RC49 (stably transfected) cell lines treated with various small molecule inhibitors.

Treatment of THP-1 cells with 80 µM Dyn2 GTPase inhibitor Dynasore inhibited CCL3 stimulated chemotaxis to sub-basal levels vs. vehicle control (p<0.001* n = 6). By comparison the Dynasore analogue Dyngo 4a, which shows higher potency as a Dyn2 GTPase inhibitor, showed a reduced ability to inhibit chemotaxis at 80 µM (48%±5 reduction in migrating cells vs. vehicle control p<0.001* n = 6). All other dynamin inhibitors (MiTMAB, OctMAB, Dynole-34-2 and Iminodyn-22) had no significant effect on chemotaxis (n = 6). The effect of Dynasore was not due to a lack of actin polymerisation (HeLa.RC49 and CHO.CCR5) or cytotoxicity (THP-1 MTS assay n = 3). The inhibition of caveolae and clathrin coated pit formation with sucrose (0.4 M) and filipin (15 µM) also had no significant effect on chemotaxis (n = 4), suggesting chemotaxis occurs independently of endocytosis. The SH3-SH2-SH3 adaptor protein GRB2 is known to bind to and activate dynamin at its C-terminal proline rich domain (PRD) via SH3: the GRB2 SH2 inhibitor CGP078850 reduced chemotaxis to sub-basal levels vs. vehicle control (<p0.001* n = 6) as did the PI3K inhibitors LY294002 (150 µM) and wortmannin (20 nM) (p<0.001* n = 6). Low concentrations of CGP078850 increased sensitivity to PI3K inhibition (n = 3) suggesting GRB2 SH2 may function upstream of Ras. The Ras inhibitor farnseylthiosalysilic acid had no significant effect on THP-1 chemotaxis at 50 µM (n = 6) indicating RAS plays no role in CCR5 induced migration.

These data suggest that a dyn2-GRB2-PI3K signalling axis is responsible for CC-chemokine mediated CTX in THP-1 and also suggests that endocytosis is not a prerequisite for chemotaxis. These signalling proteins are more commonly associated with chemotaxis signalling downstream of cytokine receptors and RTKs4. This is the first evidence for such a mechanism downstream of CCR5 and serves to broaden what is known about the functional repertoire of proteins typically associated with endocytosis and increases potential therapeutic targets for diseases caused by unwanted cell motility.

(*One way ANOVA followed by Bonferroni Multiple comparison post test.)

1. A. Mueller et al., Biochemical Pharmacology, 2006, 72, 739-748.

2. J. Zhang et al., Journal of Biological Chemistry, 1996, 271, 18302-18305.

3. M. Piehl et al., Mol. Biol. Cell, 2007, 18, 337-347.

4. K. Kawada et al., Mol. Cell. Biol., 2009, 29, 4508-4518.