title: Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK.
abstract: We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Brian S
authors.initials: BS
authors.lastname: Edwards
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: An K
authors.initials: AK
authors.lastname: Dang
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Dilyara A
authors.initials: DA
authors.lastname: Murtazina
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Melissa G
authors.initials: MG
authors.lastname: Dozier
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Whitesell
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Shaihla A
authors.initials: SA
authors.lastname: Khan
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Cherrington
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Amberg
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Colin M
authors.initials: CM
authors.lastname: Clay
authors.affiliation: Department of Zoology and Physiology (B.S.E., M.G.D., S.A.K., B.D.C., A.M.N.), University of Wyoming, Laramie, Wyoming 82071; Department of Biomedical Sciences (A.K.D., D.A.M., G.C.A., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Research and Development (J.D.W.), Allen Institute for Brain Science, Seattle, Washington 98103.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Navratil
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D022081:Cell Surface Extensions / Q000378:metabolism; D051356:Cortactin / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D052681:Gonadotrophs / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D010902:Pituitary Gland / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D034561:Sheep, Domestic
chemical_list: D000199:Actins; D020746:Calcium Channels, L-Type; D051356:Cortactin; D007987:Gonadotropin-Releasing Hormone; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D034285:Dynamin II; D034281:Dynamins; D002118:Calcium