Cytoskeletal rearrangement and Src and PI-3K-dependent Akt activation control GABA(B)R-mediated chemotaxis.
The γ-amino butyric acid (GABA) type B receptors (GABA(B)R) function as chemoattractant receptors in response to GABA(B)R agonists in human neutrophils. The goal of this study was to define signaling mechanisms regulating GABA(B)R-mediated chemotaxis and cytoskeletal rearrangement. In a proteomic study we identified serine/threonine kinase Akt, tyrosine kinases Src and Pyk2, microtubule regulator kinesin and microtubule affinity-regulating kinase (MARK) co-immunoprecipitating with GABA(B)R. To define the contributions of these candidate signaling events in GABA(B)R-mediated chemotaxis, we used rat basophilic leukemic cells (RBL-2H3 cells) stably transfected with human GABA(B1b) and GABA(B2) receptors. The GABA(B)R agonist baclofen induced Akt phosphorylation and chemotaxis by binding to its specific GABA(B)R since pretreatment of cells with CGP52432, a GABA(B)R antagonist, blocked such effects. Moreover, baclofen induced Akt phosphorylation was shown to be dependent upon PI-3K and Src kinases. Baclofen failed to stimulate actin polymerization in suspended RBL cells unless exposed to a baclofen gradient. However, baclofen stimulated both actin and tubulin polymerization in adherent RBL-GABA(B)R cells. Blockade of actin and tubulin polymerization by treatment of cells with cytochalasin D or nocodazole respectively, abolished baclofen-mediated chemotaxis. Furthermore, baclofen stimulated Pyk2 and STAT3 phosphorylation, both known regulators of cell migration. In conclusion, GABA(B)R stimulation promotes chemotaxis in RBL cells which is dependent on signaling via PI3-K/Akt, Src kinases and on rearrangement of both microtubules and actin cytoskeleton. These data define mechanisms of GABA(B)R-mediated chemotaxis which may potentially be used to therapeutically regulate cellular response to injury and disease.
Department of Medicine, University of Louisville, Louisville, KY, USA.
Michelle T
MT
Barati
Department of Medicine, University of Louisville, Louisville, KY, USA.
Janice
J
Scherzer
Department of Medicine, University of Louisville, Louisville, KY, USA.
Rui
R
Wu
Department of Medicine, University of Louisville, Louisville, KY, USA.
Madhavi J
MJ
Rane
Department of Medicine, University of Louisville, Louisville, KY, USA.|Robley Rex VA Medical Center, Zorn Avenue, Louisville, KY 40206, USA.
Jon B
JB
Klein
D008841:Actin Cytoskeleton / Q000187:drug effects; D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D058786:GABA-B Receptor Agonists / Q000494:pharmacology; D058788:GABA-B Receptor Antagonists / Q000494:pharmacology; D006801:Humans; D015739:Nocodazole / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D018080:Receptors, GABA-B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D058786:GABA-B Receptor Agonists; D058788:GABA-B Receptor Antagonists; D018080:Receptors, GABA-B; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D014404:Tubulin; D015638:Cytochalasin D; D019869:Phosphatidylinositol 3-Kinases; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D001418:Baclofen; D015739:Nocodazole
Akt; Baclofen; Chemotaxis; GABA(B) receptor; Microtubules; Src