title: Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner.
abstract: U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.
authors.affiliation: Division of Allergy and Immunology, The Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Meshki
authors.forename: Steven D
authors.initials: SD
authors.lastname: Douglas
authors.forename: Mingyue
authors.initials: M
authors.lastname: Hu
authors.forename: Susan E
authors.initials: SE
authors.lastname: Leeman
authors.forename: Florin
authors.initials: F
authors.lastname: Tuluc
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013373:Substance P / Q000494:pharmacology*; D014404:Tubulin / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D014404:Tubulin; D049452:Green Fluorescent Proteins; D013373:Substance P; D054462:p21-Activated Kinases; D003078:Colchicine; D002118:Calcium