M3 muscarinic acetylcholine receptor dysfunction inhibits Rac1 activity and disrupts VE-cadherin/β-catenin and actin cytoskeleton interaction.
The objective was to investigate whether M3 muscarinic acetylcholine receptor (mAChR) dysfunction disrupts the linkage between the vascular endothelial (VE)-cadherin in the adherens junctional complex and the actin-based cytoskeleton, increasing vascular permeability in atherosclerosis. Western blotting revealed that a selective M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and M3 receptor siRNA decrease VE-cadherin and β-catenin in Triton X-100-insoluble fractions, indicating that M3 receptor inhibition weakens the linkage between the VE-cadherin/β-catenin complex and the actin cytoskeleton. Co-immunoprecipitation assays showed that M3 receptor inhibition reduces Rac1 activity and the association of IQ motif-containing GTPase-activating protein 1 (IQGAP1) with Ras-related C3 botulinum toxin substrate 1 (Rac1), while increasing the interaction between IQGAP1 and β-catenin. Using IQGAP1 siRNA, we found that IQGAP1 is required for stable interaction between VE-cadherin/β-catenin and the actin cytoskeleton in quiescent endothelial cells; IQGAP1 siRNA augments the M3 receptor inhibition-induced dissociation between them. Moreover, S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, attenuates this disassociation and Rac1 activity inhibition. The M3 receptor facilitates interaction of the VE-cadherin-based adherens junctional complex and the actin-based cytoskeleton by maintaining Rac1 activity, which regulates the interaction between IQGAP1/Rac1 and IQGAP1/β-catenin, and may contribute to endothelial barrier function under physiological conditions.
a Department of Cardiovascular Surgery, Xiehe Hospital, Huazhong University of Science and Technology, Wuhan, 430022, China.
Zhou-Yang
ZY
Jiao
Jing
J
Wu
Bing
B
Wen
Wen-Zeng
WZ
Zhao
Xin-Ling
XL
Du
D008841:Actin Cytoskeleton / Q000378:metabolism*; D022005:Adherens Junctions / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D009569:Nitric Oxide; D043587:Receptor, Muscarinic M3; D051176:beta Catenin; D020830:rac1 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins; D000099152:Cadherin 5; C101203:IQ motif containing GTPase activating protein 1; C107953:RAC1 protein, human