WNK4 kinase negatively regulates the surface expression of muscarinic M3 receptor.
With-No-Lysine [K] 4 (WNK4) kinase regulates the surface expression of various ion transporters. Not only ion transporters but G-protein coupled receptors (GPCR) can function properly when their expression level is appropriate at the plasma membrane. In this study, we examined the role of WNK4 kinase in the regulation of muscarinic receptor 3 (M₃R) using physiological and biochemical experiments. Measurement of the pilocarpine responsive [Ca(2+)](i) change demonstrated that WNK4 kinase decreased the activity of M(3)R through its reduced surface expression. Kinase domain of WNK4 bound with the third intracellular region of M₃R whereas its negative regulation was independent on the kinase activity. Comparable to wild-type WNK4, kinase-inactive WNK4(D318A) mutant also reduced the surface expression of M(3)R, whereas the kinase domain of WNK4₁₋₄₄₁ failed to reduce the surface expression of M₃R. In accordance with surface biotinylation experiments, non-permeable immunostaining of M₃R also showed that M₃R surface expression is independent on the kinase activity of WNK4. Interestingly, comparison of the half life of total and surface M₃R revealed that only the half life of total M₃R, but not surface M₃R was decreased by WNK4 kinase. Nevertheless, the rate of decrease in surface M₃R always exceeded that of total M₃R. Taken together, these results suggest that WNK4 kinase negatively regulates the anterograde trafficking of M₃R through kinase-independent mechanism.
Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
Woo Young
WY
Chung
Hyun Woo
HW
Park
Dae Keon
DK
Heo
Kyung Hwan
KH
Kim
Min Goo
MG
Lee
Joo Young
JY
Kim
D020013:Calcium Signaling; D015536:Down-Regulation; D006801:Humans; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D021381:Protein Transport; D043587:Receptor, Muscarinic M3 / Q000737:chemistry / Q000378:metabolism*
D043587:Receptor, Muscarinic M3; D017346:Protein Serine-Threonine Kinases; C433806:WNK4 protein, human