The monomeric G-proteins Rac1 and/or Cdc42 are required for the inhibition of voltage-dependent calcium current by bradykinin.
Although regulation of voltage-dependent calcium current (ICa,V) by neurotransmitters is a ubiquitous mechanism among nerve cells, the signaling pathways involved are not well understood. We have determined previously that in a neuroblastoma-glioma hybrid cell line (NG108-15), the heterotrimeric G-protein G13 mediates the inhibition of ICa,V produced by bradykinin (BK) via an unknown mechanism. Various reports indicate that G13 can couple to RhoA, Rac1, and Cdc42, which are closely related members of the Rho family of monomeric G-proteins. We have investigated their role as signaling intermediates in the pathway used by BK to inhibit ICa,V. Using immunoblot analysis and the PCR, we found evidence that RhoA, Rac1, and Cdc42 all are expressed in NG108-15 cells. Intracellularly perfused recombinant Rho-GDI (an inhibitor of guanine nucleotide exchange specific for the Rho family) attenuated the inhibition of ICa,V by BK. These findings indicate that activation of RhoA, Rac1, or Cdc42 may be required for the response to BK. To determine whether any of these monomeric G-proteins mediate the response to BK, we have intracellularly applied blocking antibodies specific for each of the candidate proteins. Only the anti-Rac1 antibody blocked the response to BK. In parallel experiments, peptides corresponding to the C-terminal regions of Rac1 and Cdc42 blocked the same response. These data indicate a novel functional contribution of Rac1 and possibly also of Cdc42 to the inhibition of ICa,V by neurotransmitters.
Wilk-Blaszczak
M A
MA
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Singer
W D
WD
Quill
T
T
Miller
B
B
Frost
J A
JA
Sternweis
P C
PC
Belardetti
F
F
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001667:Binding, Competitive / Q000502:physiology; D001920:Bradykinin / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000378:metabolism*; D004558:Electric Stimulation; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D005910:Glioma; D020730:Guanine Nucleotide Dissociation Inhibitors; D006822:Hybrid Cells / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D015151:Immunoblotting; D015640:Ion Channel Gating / Q000502:physiology; D051379:Mice; D009447:Neuroblastoma; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein
D000906:Antibodies; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018797:Cell Cycle Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D010446:Peptide Fragments; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D001920:Bradykinin