title: Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components.
abstract: Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Head
authors.forename: Hemal H
authors.initials: HH
authors.lastname: Patel
authors.forename: David M
authors.initials: DM
authors.lastname: Roth
authors.forename: Fiona
authors.initials: F
authors.lastname: Murray
authors.forename: James S
authors.initials: JS
authors.lastname: Swaney
authors.forename: Ingrid R
authors.initials: IR
authors.lastname: Niesman
authors.forename: Marilyn G
authors.initials: MG
authors.lastname: Farquhar
authors.forename: Paul A
authors.initials: PA
authors.lastname: Insel
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D022461:Caveolins / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D003078:Colchicine / Q000378:metabolism; D003285:Contractile Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D015638:Cytochalasin D / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D064448:Filamins; D007545:Isoproterenol / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000648:ultrastructure; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism / Q000648:ultrastructure; D015398:Signal Transduction / Q000502:physiology*; D050257:Tubulin Modulators / Q000378:metabolism
chemical_list: D000199:Actins; D000318:Adrenergic beta-Agonists; D022461:Caveolins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D020033:Protein Isoforms; D011943:Receptors, Adrenergic, beta; D043562:Receptors, G-Protein-Coupled; D050257:Tubulin Modulators; D015638:Cytochalasin D; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D003078:Colchicine