The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity.
The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.
Monastyrskaya
Katia
K
Department of Cell Biology, Institute of Anatomy, University of Bern, Switzerland. monastyk@ana.unibe.ch
Hostettler
Andrea
A
Buergi
Sibylle
S
Draeger
Annette
A
D000818:Animals; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D010766:Phosphorylation; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013373:Substance P / Q000502:physiology; D013552:Swine; D014162:Transfection
D018040:Receptors, Neurokinin-1; D013373:Substance P; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3