title: Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay.
abstract: Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.
authors.affiliation: Laboratory of Immunogenetics Imaging Facility, NIAID/NIH, Rockville, MD 20852, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Brzostowski
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sawai
authors.forename: Orr
authors.initials: O
authors.lastname: Rozov
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Liao
authors.forename: Daisuke
authors.initials: D
authors.lastname: Imoto
authors.forename: Carole A
authors.initials: CA
authors.lastname: Parent
authors.forename: Alan R
authors.initials: AR
authors.lastname: Kimmel
mesh_terms: D000199:Actins / Q000378:metabolism*; D002478:Cells, Cultured; D002633:Chemotaxis / Q000502:physiology*; D004023:Dictyostelium / Q000166:cytology / Q000378:metabolism*; D000076225:Mechanistic Target of Rapamycin Complex 2; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015800:Protozoan Proteins / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D000076225:Mechanistic Target of Rapamycin Complex 2; D019950:Mitogen-Activated Protein Kinase 1; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D015800:Protozoan Proteins; D011953:Receptors, Cyclic AMP; D043562:Receptors, G-Protein-Coupled; D058570:TOR Serine-Threonine Kinases; C068125:cyclic AMP receptor cAR1
keywords: AKT; ERK2; GPCR; Heterotrimeric G proteins; Oscillations; PI3K; TORC2; cAMP
references.citation: Eukaryot Cell. 2003 Feb;2(1):62-75
references.pmid: 12582123
references.citation: J Biol Chem. 2006 Nov 24;281(47):35931-41
references.pmid: 16990258
references.citation: PLoS One. 2010 Dec 13;5(12):e15249
references.pmid: 21179207
references.citation: J Biol Chem. 2002 Aug 30;277(35):31567-76
references.pmid: 12077128
references.citation: Annu Rev Biophys. 2010;39:265-89
references.pmid: 20192768
references.citation: Cancer Res. 2006 Jun 1;66(11):5665-75
references.pmid: 16740704
references.citation: Sci Signal. 2012 Jan 03;5(205):ra2
references.pmid: 22215733
references.citation: J Biol Chem. 1995 Apr 14;270(15):8667-72
references.pmid: 7721769
references.citation: Mol Biol Cell. 2000 Aug;11(8):2803-20
references.pmid: 10930471
references.citation: EMBO J. 2005 Jul 6;24(13):2254-64
references.pmid: 15962002
references.citation: Trends Cell Biol. 2009 Oct;19(10):523-30
references.pmid: 19733079
references.citation: Curr Biol. 2002 Jul 23;12(14):1199-208
references.pmid: 12176329
references.citation: Dev Biol. 1986 Apr;114(2):529-33
references.pmid: 3007244
references.citation: Mol Biol Cell. 2006 Oct;17(10):4220-7
references.pmid: 16870702
references.citation: Dev Growth Differ. 2011 May;53(4):503-17
references.pmid: 21585355
references.citation: J Biol Chem. 1994 Mar 4;269(9):7036-44
references.pmid: 8120068
references.citation: Mol Biol Cell. 2006 Jan;17(1):357-66
references.pmid: 16267269
references.citation: Mol Pharmacol. 2011 Nov;80(5):760-8
references.pmid: 21836019
references.citation: Trends Pharmacol Sci. 2011 Sep;32(9):521-33
references.pmid: 21680031
references.citation: Science. 2004 May 7;304(5672):875-8
references.pmid: 15131307
references.citation: Pharmacol Rev. 2001 Mar;53(1):1-24
references.pmid: 11171937
references.citation: Genome Biol. 2007;8(7):R144
references.pmid: 17659086
references.citation: Mol Biol Cell. 2005 Oct;16(10):4572-83
references.pmid: 16079174
references.citation: J Biol Chem. 1986 Nov 15;261(32):15121-5
references.pmid: 3771567
references.citation: Cell Signal. 2009 Jul;21(7):1045-53
references.pmid: 19249352
references.citation: Dev Cell. 2012 Jan 17;22(1):92-103
references.pmid: 22264729
references.citation: J Cell Biol. 1994 Sep;126(6):1537-45
references.pmid: 8089184
references.citation: Nat Protoc. 2011 Apr;6(4):427-38
references.pmid: 21412271
references.citation: Annu Rev Physiol. 2007;69:535-60
references.pmid: 17002593
references.citation: Annu Rev Biochem. 1998;67:653-92
references.pmid: 9759500
references.citation: J Leukoc Biol. 2006 Dec;80(6):1214-21
references.pmid: 16943386
references.citation: J Gen Microbiol. 1974 Dec;85(2):321-34
references.pmid: 4615133
references.citation: Science. 1981 Apr 24;212(4493):443-6
references.pmid: 6259734
references.citation: Methods Mol Biol. 2006;346:281-96
references.pmid: 16957297
references.citation: Ageing Res Rev. 2008 Jul;7(3):234-48
references.pmid: 18657484
references.citation: J Cell Sci. 2010 Mar 15;123(Pt 6):983-92
references.pmid: 20200230
references.citation: J Cell Biol. 1982 Mar;92(3):807-21
references.pmid: 6282894
references.citation: J Biol Chem. 2011 Sep 16;286(37):32188-97
references.pmid: 21730065
references.citation: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7478-83
references.pmid: 12032308
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9649-54
references.pmid: 19497875
references.citation: J Biol Chem. 1981 Jul 25;256(14):7620-7
references.pmid: 6265455
references.citation: Curr Biol. 2004 Jan 6;14(1):1-10
references.pmid: 14711408
references.citation: J Biol Chem. 2004 Sep 3;279(36):37215-8
references.pmid: 15231839
references.citation: Nature. 2005 Jan 20;433(7023):323-6
references.pmid: 15662425
references.citation: J Immunol. 2005 Oct 15;175(8):5396-402
references.pmid: 16210646
references.citation: J Cell Biol. 1987 Nov;105(5):2301-6
references.pmid: 2890646
references.citation: Trends Biochem Sci. 2011 Sep;36(9):457-69
references.pmid: 21764321
references.citation: Science. 2010 May 21;328(5981):1021-5
references.pmid: 20413456
references.citation: Immunol Res. 2009;43(1-3):118-27
references.pmid: 18827980
references.citation: Dev Biol. 1984 Jan;101(1):136-46
references.pmid: 6319207
references.citation: Science. 2001 Mar 23;291(5512):2408-11
references.pmid: 11264536
references.citation: J Biol Chem. 1997 Oct 24;272(43):27313-8
references.pmid: 9341180
references.citation: Eukaryot Cell. 2009 Sep;8(9):1381-96
references.pmid: 19633268
references.citation: Dev Biol. 2001 May 1;233(1):225-36
references.pmid: 11319871
references.citation: J Immunol Methods. 2007 Mar 30;320(1-2):155-63
references.pmid: 17289072
references.citation: J Immunol. 2003 Dec 1;171(11):6128-34
references.pmid: 14634128
references.citation: Trends Pharmacol Sci. 2012 Mar;33(3):154-64
references.pmid: 22277298
references.citation: Cell. 1998 Oct 2;95(1):81-91
references.pmid: 9778249
references.citation: Integr Biol (Camb). 2011 Jun;3(6):675-83
references.pmid: 21541374
references.citation: J Leukoc Biol. 2004 Apr;75(4):698-704
references.pmid: 14704365
references.citation: Dev Genet. 1991;12(1-2):6-13
references.pmid: 2049880
references.citation: Development. 2011 Feb;138(3):387-96
references.pmid: 21205784
references.citation: J Cell Sci. 1976 Dec;22(3):645-56
references.pmid: 1035221
references.citation: J Biol Chem. 1988 Oct 5;263(28):14538-43
references.pmid: 2844774
references.citation: Mol Biol Cell. 2005 Feb;16(2):676-88
references.pmid: 15563608
references.citation: Biophys J. 2004 Dec;87(6):3764-74
references.pmid: 15465874
references.citation: Curr Biol. 1998 Mar 26;8(7):405-8
references.pmid: 9545201
references.citation: Genes Dev. 2004 Apr 1;18(7):805-15
references.pmid: 15059962
references.citation: J Biol Chem. 2001 Sep 14;276(37):34408-18
references.pmid: 11448957
references.citation: J Biol Chem. 1985 Feb 10;260(3):1757-64
references.pmid: 2981872
references.citation: J Biol Chem. 1994 Jan 14;269(2):1523-32
references.pmid: 8288619
references.citation: Mol Biol Cell. 1994 Jun;5(6):703-11
references.pmid: 7949426
references.citation: J Biol Chem. 1987 Jun 5;262(16):7700-4
references.pmid: 3034902