The NK1 Receptor Localizes to the Plasma Membrane Microdomains, and Its Activation Is Dependent on Lipid Raft Integrity

Abstract

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 β2-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-β-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. 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 β2-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-β-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. The plasma membrane is segregated into domains, which incorporate specific lipid and protein moieties. These lateral assemblies of cholesterol and glycosphingolipids form dynamic structures termed lipid “rafts” or microdomains, and are associated with characteristic sets of proteins, linked by glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; GPCR, G protein-coupled receptor; PLC, phospholipase C; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; SM, smooth muscle; NK1R, neurokinin 1 receptor; MBCD, methyl-β-cyclodextrin; PMA, 4β-phorbol 12-myristate 13-acetate; HEK, human embryonic kidney cells; Ab, antibody; SP, substance P. anchors to their extracellular face and/or multiple acylated cytoplasmic proteins to the inside of the cell. Membrane rafts can self-associate to form higher order structures with higher physical stability and constitute hubs of signaling activity (1Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2060) Google Scholar). Over the last few years, a wide array of signaling and receptor proteins have been found to associate with lipid rafts, which further strengthened this concept (2Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: 843-851PubMed Google Scholar). Signal transduction across the plasma membrane is a fundamental aspect of cellular communication in which G protein-coupled receptors (GPCRs) play a major role. Molecular regulation by these seven transmembrane receptors results in the transmission of an extracellular signal from the receptors via heterotrimeric (α-, β-, and γ-subunit-containing) G proteins to effector molecules. Each cell expresses a number of different GPCRs, which may share their downstream signaling partners. Since GPCRs remain major targets for pharmacological manipulation, their activation and blockade by specific agonists and antagonists have been extensively investigated. Most of this work has been aimed at studying the receptor-ligand interaction, disregarding the downstream events of GPCR signaling, yet many G protein-coupled receptors have the capacity to interact with multiple G proteins and thus regulate more than one effector pathway (3Chabre O. Conklin B.R. Brandon S. Bourne H.R. Limbird L.E. J. Biol. Chem. 1994; 269: 5730-5734Abstract Full Text PDF PubMed Google Scholar). Such observations contradict the paradigm that receptors and other integral membrane proteins are randomly distributed in the plasma membrane. Indeed, fluorescence labeling experiments have demonstrated that the distribution of G proteins is not random and that both βγ- and α-subunits display limited mobility (4Fishburn C.S. Pollitt S.K. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1085-1090Crossref PubMed Scopus (51) Google Scholar). Since the receptors themselves interact with various scaffolding molecules, (5Edwards S.W. Tan C.M. Limbird L.E. Trends Pharmacol. Sci. 2000; 21: 304-308Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), it is highly likely that GPCRs form a part of the pre-assembled and relatively stable signal transduction complexes within the plasma membrane. G protein-coupled receptors play an important role in the regulation of smooth muscle (SM) contraction and relaxation, as well as other important processes such as growth, remodeling, and inflammation (6Billington C.K. Penn R.B. Respir. Res. 2003; 4: 2Crossref PubMed Google Scholar). A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle (7Ostrom R.S. Liu X. Head B.P. Gregorian C. Seasholtz T.M. Insel P.A. Mol. Pharmacol. 2002; 62: 983-992Crossref PubMed Scopus (123) Google Scholar). β-Adrenergic receptors are the primary therapeutic targets in airways since their activation promotes relaxation of airway smooth muscle and combats acute asthma attacks. The Gq-coupled receptors, on the other hand, are the principal mediators of SM contraction. They activate PLC, which causes an increase of the intracellular Ca2+ through IP3-mediated release from sarcoplasmic calcium stores. The raise in Ca2+ promotes to activate and the and contraction. The is that many components of the GPCR signal transduction in muscle cells are to specific membrane and P. Mol. Biol. PubMed Google have that of G proteins in and lipid rafts. with the of G proteins in rafts, of the GPCR as well as proteins, PLC, and activated PKC have been to to rafts and X. Res. PubMed Google X. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Res. Scopus Google J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). localization is a dynamic as it has been that the β2-adrenergic receptor receptor and receptor that are distributed the plasma membrane to in the of O. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). receptors to and are expressed in the central and peripheral nervous as well as in the and NK1 receptor, which has Substance as its smooth muscle and secretion, and 1998; PubMed Scopus Google Scholar). NK1 has been extensively its membrane signal and distribution A. J. Pharmacol. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). of the of the signaling is its to to different signaling which and intracellular Ca2+ and to activation of and of Using and of protein U. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have that the pharmacological of this receptor, which at cell is a of complexes of NK1 with or and that these complexes not on this the the of NK1 receptors in different membrane We investigated the plasma membrane localization of the NK1 receptor, and show that this receptor is to lipid rafts. of rafts cholesterol extraction with to the of the NK1R signaling complexes and receptor of to the NK1R causes receptor and protein kinase C which in the signaling by the receptor O. Am. J. Physiol. Physiol. PubMed Google Scholar). We show that activation of the NK1R of PKC into lipid rafts, it with the receptor and causes its signal-regulated 1 and are to the downstream effectors of the NK1R signaling O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Google O. S. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. 2003; PubMed Scopus Google Scholar). we the of lipid on the NK1R-mediated activation of and show that the not in cells the and and from NK1R, and from as well as MBCD, and 4β-phorbol 12-myristate from and and the from and from was from Molecular The was from and NK1 receptor was from and and HepG2 as well as human smooth muscle cell primary and human smooth muscle in of of cells in at in a HEK293 and HepG2 cells transiently with using at for and by and with which NK1R used of the cells with and was as described J. U. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). using the cells and with 1 and and for 1 at with the primary in the and with the using the the plasma membrane of the cells was for with A with Molecular the cells and with with by a primary in a of and of and Protein rafts by extraction on and on of or and on in using cells the and by of of the on in the of and the of the was to by the of in A was the The for at in at from the of the and by was to their protein of the was by the lipid rafts found to at the to and of the of cells NK1R the and in of A on and to The through and at The with 1 of with at for and in of the Protein was and to across the of by and in was using as a as described previously J. U. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). rafts by in and of for the Protein in was using as described by the to the of protein into activity in was as which was at a of was expressed as an at of the of experiments receptor signaling was by Ca2+ calcium the cells NK1R with for at at on a and using the and increase in fluorescence of to the was after the of experiments is of was by NK1R to for at by in the and of by and with The of the NK1R, and the protein the across the as by with as well as the with expressed as increase to cells in of cell and of and in at for was in the of the was by 1 of 1 of the was through with and the with of into and after of The protein of the cell was by protein was as the in the of and in the of and expressed as by using and and and as described previously S. A. J. 2002; PubMed Scopus Google Scholar). Protein of the and was using of the NK1 in and in NK1R was expressed in its from human with as described previously A. E. P. J. 1994; PubMed Google Scholar). The NK1R was with the at the using the and the been to have effect on the receptor signaling and localization J. A. J. 1994; PubMed Google Scholar). The receptor was transiently expressed in HEK293 and HepG2 cells using and its and plasma membrane localization by and with both and and as well as and smooth muscle primary cell for the of the We found that and as well as with in Our of not the of the NK1R in was further by these cells with Substance and the intracellular Ca2+ release by calcium of to by of not The that not the cells to SP, a relatively of the NK1R as by and can by of the smooth muscle cells in with of of of the NK1R signal transduction pathway The NK1R to the Membrane rafts extraction and on as described previously J. PubMed Scopus Google Scholar). Since has an important effect on the of extraction of from to and by the of proteins and extraction of the of the on in the of lipid rafts by in as described previously A. Biol. 2003; PubMed Scopus Google Scholar). Our earlier experiments S. A. J. 2002; PubMed Scopus Google demonstrated that lipid rafts using extraction with on for the proteins 1 and with the of raft-associated proteins and and at the and of the The NK1 receptor in and and in the to the and of the The and the similar localization The NK1R, expressed in the HEK293 cells to the extraction of lipid microdomains and on of the receptor with not the of the receptor from lipid rafts, was a of the of NK1R into the and which of the NK1R its Mol. Biol. PubMed Google Scholar). Our of lipid not to and rafts. order to whether the of is for the NK1R with rafts, we of the into HepG2 of this protein A. R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We found similar to the HEK293 in HepG2 cells the NK1R, the receptor to microdomains, in the as the raft-associated proteins and with was not for the lipid targeting of the The extraction and experiments used not to determine the of the NK1R with the other raft-associated Indeed, the that of the form at different than NK1R a of the of the in the of distribution of in which are not integral membrane proteins and with lipid rafts on such as The NK1R and Its to the NK1 receptor, or to for to activate to Ca2+ or Substance to activate the NK1 the lipid rafts and the of in the by and microdomains in the and as using as a the of to the and activation by or Ca2+ increase its with the lipid rafts in and in with the previously observations in X. Res. PubMed Google Scholar). Ca2+ by NK1 signaling to in of into rafts of PKC with signaling by GPCR, the NK1R O. Am. J. Physiol. Physiol. PubMed Google J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the activated to lipid rafts important for the effect of on Ca2+ in HEK293 HEK293 cells the NK1R with to the intracellular Ca2+ the cells with to activate PKC, their to activate the intracellular Ca2+ release was PKC the of the NK1R, it from the G proteins O. Am. J. Physiol. Physiol. PubMed Google Scholar). is that of the activated PKC into the NK1R is this NK1R by with MBCD, and by of cholesterol from cells with to the of lipid rafts and of their The of themselves in a of raft-associated molecules, the NK1R, from the of cholesterol in the can by the cells with an the lipid microdomains and their protein HEK293 cells the NK1 receptor cholesterol with for or extraction for 1 with to cholesterol rafts as described and their protein by with the NK1R and the raft-associated proteins and extraction was by and its using extraction of the of in the and at the and of of the NK1R, and the other raft-associated and from the into the and of cholesterol for 1 its in the plasma membrane and at the lipid rafts as by the protein of the and of the NK1R to Substance of rafts as pre-assembled signaling we to determine whether the and of the NK1R to SP, by intracellular calcium is the of rafts by HEK293 cells the NK1R with and the Ca2+ by Ca2+ cells to of of the cells with for at to and to at the to O. Am. J. Physiol. Physiol. PubMed Google signaling was the cells to the at a higher and with a of can that the not the of the receptor to to its down-stream effectors, it the receptor to by its coupling to which is in lipid rafts. of not receptor or its to interact with its since of by of the not in of the NK1R-mediated Ca2+ order to that of the NK1R-mediated signaling was by of lipid and not by of cholesterol we these experiments using and cholesterol within the plasma complexes that Res. PubMed Scopus (70) Google Scholar). The cells with and with for 1 to of rafts by similar to cholesterol extraction by MBCD, the of NK1R of more at of SP, than after at and SP, cells of the These of lipid which has been by the Res. PubMed Scopus (70) Google Scholar). 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A. 1998; PubMed Scopus Google Scholar). its to associate with rafts of its and not an of a system used in these Upon of substance to the cells to lipid was a of the NK1R distribution into the which of receptor the cells after of the and experiments this further with was as for the and extraction of the membrane proteins and 1 as well as lipid as demonstrated the localization of the NK1R and its effector we to determine whether downstream NK1R signaling events involved these plasma membrane of by GPCRs and intracellular Ca2+ release to activation of the kinase which in its in the translocates to the plasma membrane and has been to associate with X. Res. PubMed Google Scholar). we show that stimulation of the NK1R cells with causes to from the of the to the activation by or Ca2+ the A of the of PKC and the NK1R in the plasma membrane is the of receptor signaling to SP, which was after of cells with and is by receptor O. Am. J. Physiol. Physiol. PubMed Google Scholar). of cholesterol with to of rafts and is used to of the cells with for 1 the rafts and the NK1R as well as the other raft-associated proteins with and in the and in the abolished the NK1R signaling as by Ca2+ in to of that the of the receptor was for its coupling with is similar to the NK1R, from the and not the of MBCD, was for receptor since a higher of the the signaling the acute of the which and cholesterol within the plasma membrane and thus lipid rafts, similar as in the NK1R of cholesterol for 1 after extraction the lipid and protein of the rafts, and the The of as well as the that of cholesterol by not the of the NK1R at the plasma membrane or the that of the NK1R with its effectors within lipid rafts is important for the of the NK1R to GPCRs and receptor activation are by of signaling, in order to which to the receptor of of signal transduction by of the to different agonists and of receptors from the plasma membrane by by of receptors from the G proteins receptor by G protein receptor and of the NK1 receptor not and that the NK1 receptor by and by in and play a role in the of the receptors from the G proteins and to the for PubMed Google Scholar). 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Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), has been to associated both with and and in and J. Biol. 2003; PubMed Scopus Google J. Sci. 2003; PubMed Scopus Google Scholar). is that the spatial of the NK1R and its and which is by their in lipid rafts, is essential for coupling of the NK1R signaling and activation. The results that lipid microdomains are essential for coupling of the NK1 receptor to and activation of the signaling which to intracellular calcium recruitment of PKC of this work we not the and rafts, that the of was not a for the NK1R with rafts. into the of the NK1R to to the as well as the pathway S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), it is that in cells are to the signaling of their to and and P. Mol. Biol. PubMed Google Scholar). further the as a of the importance of membrane microarchitecture for NK1 We E. and U. for and the C. of for and S. for with