WNK4 kinase negatively regulates the surface expression of muscarinic M3 receptor

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• With-No-Lysine [K] 4 (WNK4) kinase is crucial for regulating the plasma membrane expression of ion transporters.
• Proper cell surface expression of G-protein coupled receptors (GPCRs), like muscarinic receptor 3 (M₃R), is essential for their function.

Purpose of the Study:

• To investigate the role of WNK4 kinase in modulating the surface expression and activity of muscarinic receptor 3 (M₃R).
• To elucidate the mechanism by which WNK4 kinase affects M₃R trafficking and function.

Main Methods:

• Physiological experiments measuring pilocarpine-induced intracellular calcium ([Ca(2+)](i)) changes.
• Biochemical assays including surface biotinylation and immunostaining.
• Analysis of M₃R total and surface protein half-life.
• Utilized wild-type and kinase-inactive WNK4 mutants (WNK4(D318A)) and WNK4 fragments.

Main Results:

• WNK4 kinase significantly reduces M₃R activity, correlating with decreased surface expression.
• The interaction between WNK4 kinase domain and M₃R's third intracellular region is key, but regulation is independent of WNK4's kinase activity.
• Both wild-type and kinase-inactive WNK4 mutants reduced M₃R surface expression, while a WNK4 fragment lacking the kinase domain did not.
• WNK4 decreases the half-life of total M₃R but not surface M₃R, indicating accelerated degradation of total M₃R.
• The rate of decrease in surface M₃R exceeded that of total M₃R, suggesting impaired anterograde trafficking.

Conclusions:

• WNK4 kinase negatively regulates M₃R surface expression and activity through a kinase-independent mechanism.
• WNK4 appears to inhibit the anterograde trafficking of M₃R to the plasma membrane.
• These findings highlight a novel regulatory pathway for GPCR surface expression involving WNK4.