NEUROMEDIN-B RECEPTOR ACTIVATION CAUSES TYROSINE PHOSPHORYLATION OF P125(FAK) BY A PHOSPHOLIPASE-C INDEPENDENT MECHANISM WHICH REQUIRES P21(RHO) AND INTEGRITY OF THE ACTIN CYTOSKELETON

Recent studies show that tyrosine phosphorylation by a number of neuro peptides may be an important intracellular pathway in mediating change s in cell function, particularly related to growth. Neuromedin B (NMB) , a mammalian bombesin related peptide, functions through a distinct r eceptor, the neuromedin B receptor (NMB-R), of which little is known a bout its cellular basis of action. In the present study we explored th e ability of NMB-R activation to cause tyrosine phosphorylation of foc al adhesion kinase (p125(FAK)), an important substrate for tyrosine ph osphorylation by other neuropeptides. NMB caused rapid increases in p1 25(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B re ceptor (rNMR-R) transfected BALE 3T3 cells. NMB had a half-maximal eff ect was at 0.4 nM and was 30-fold more potent than gastrin-releasing p eptide (GRP). The stoichiometric relationships between increased p125( FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 mu M) caused 45 % and the calcium ionophore, A23187, 11% of maximal tyrosine phosphory lation of p125(FAK) seen with NMB. A23187 potentiated the effect of TP A. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhi bited NMB-stimulated increases in [Ca2+](i), but had no effect on NMB- stimulation of p125(FAK) phosphorylation either alone or with GF109203 X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induce d phosphorylation of p125(FAK) by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [H-3]inositol phosphates. Cytocha lasin D, an agent which disrupts actin microfilaments, inhibited BN-an d TPA-induced tyrosine phosphorylation of p125(FAK) completely. In con trast, colchicine, an agent which disrupts microtubules, had no effect . Pretreatment with Clostridium botulinum C3 exoenzyme which inactivat es the small GTP-binding protein rho p21, also inhibited tyrosine phos phorylation of p125(FAK) by 55%. These results demonstrate that activa tion of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). N MB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB -induced changes in [Ca2+](i) or PKC. The integrity of the actin cytos keleton but not of microtubules is necessary for NMB-stimulated phosph orylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine p hosphorylation of p125(FAK). These results suggest that similar to som e other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growt h.