title: Multiple regulatory roles of the carboxy terminus of Ste2p a yeast GPCR.
abstract: Signaling and internalization of Ste2p, a model G protein-coupled receptor (GPCR) from the yeast Saccharomyces cerevisiae, are reported to be regulated by phosphorylation status of serine (S) and threonine (T) residues located in the cytoplasmic C-terminus. Although the functional roles of S/T residues located in certain C-terminus regions are relatively well characterized, systemic analyses have not been conducted for all the S/T residues that are spread throughout the C-terminus. A point mutation to alanine was introduced into the S/T residues located within three intracellular loops and the C-terminus individually or in combination. A series of functional assays such as internalization, FUS1-lacZ induction, and growth arrest were conducted in comparison between WT- and mutant Ste2p. The Ste2p in which all S/T residues in the C-terminus were mutated to alanine was more sensitive to α-factor, suggesting that phosphorylation in the C-terminus exerts negative regulatory activities on the Ste2p signaling. C-terminal S/T residues proximal to the seventh transmembrane domain were important for ligand-induced G protein coupling but not for receptor internalization. Sites on the central region of the C-terminus regulated both constitutive and ligand-induced internalization. Residues on the distal part were important for constitutive desensitization and modulated the G protein signaling mediated through the proximal part of the C-terminus. This study demonstrated that the C-terminus contains multiple functional domains with differential and interdependent roles in regulating Ste2p function in which the S/T residues located in each domain play critical roles.
authors.affiliation: Department of Pharmacology, College of Pharmacy, Chonnam National University, Kwang-Ju 500-757, Republic of Korea.
authors.forename: Kyeong-Man
authors.initials: KM
authors.lastname: Kim
authors.forename: Yong-Hun
authors.initials: YH
authors.lastname: Lee
authors.forename: Ayca
authors.initials: A
authors.lastname: Akal-Strader
authors.forename: M Seraj
authors.initials: MS
authors.lastname: Uddin
authors.forename: Melinda
authors.initials: M
authors.lastname: Hauser
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Becker
mesh_terms: D000595:Amino Acid Sequence; D004705:Endocytosis; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005838:Genotype; D007763:Lac Operon; D008024:Ligands; D000072235:Mating Factor; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D051964:Receptors, Mating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D015398:Signal Transduction; D013912:Threonine; D013997:Time Factors; D015854:Up-Regulation
chemical_list: C490024:FUS1 protein, S cerevisiae; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D012694:Serine; D000072235:Mating Factor
references.citation: J Cell Biol. 1998 Apr 20;141(2):349-58
references.pmid: 9548714
references.citation: J Biol Chem. 2006 Jan 27;281(4):2263-72
references.pmid: 16314417
references.citation: Trends Pharmacol Sci. 2008 Aug;29(8):413-20
references.pmid: 18606460
references.citation: Gene. 1995 Apr 14;156(1):119-22
references.pmid: 7737504
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2193-8
references.pmid: 17287358
references.citation: Annu Rev Pharmacol Toxicol. 1998;38:289-319
references.pmid: 9597157
references.citation: Nat Biotechnol. 2008 Dec;26(12):1339-40
references.pmid: 19060867
references.citation: Mol Biol Cell. 1993 May;4(5):511-21
references.pmid: 8392878
references.citation: Methods Enzymol. 2002;344:617-31
references.pmid: 11771415
references.citation: Nature. 2008 Jul 24;454(7203):486-91
references.pmid: 18594507
references.citation: J Biol Chem. 1988 Nov 25;263(33):17333-41
references.pmid: 2846561
references.citation: J Biol Chem. 1997 Jun 13;272(24):15553-61
references.pmid: 9182592
references.citation: Mol Cell Biol. 1996 Jan;16(1):247-57
references.pmid: 8524302
references.citation: Cell. 1988 Oct 21;55(2):221-34
references.pmid: 2844413
references.citation: J Biol Chem. 1994 Jan 14;269(2):968-73
references.pmid: 8288649
references.citation: Trends Pharmacol Sci. 1990 May;11(5):190-4
references.pmid: 2188401
references.citation: Cell. 1988 Aug 26;54(5):609-20
references.pmid: 2842059
references.citation: J Biol Chem. 1995 Oct 20;270(42):24782-9
references.pmid: 7559596
references.citation: J Biol Chem. 2006 Feb 17;281(7):4109-16
references.pmid: 16354660
references.citation: J Biol Chem. 2002 Aug 23;277(34):30581-90
references.pmid: 12058045
references.citation: Biochemistry. 2005 Sep 6;44(35):11795-810
references.pmid: 16128581
references.citation: Nature. 2007 Nov 15;450(7168):383-7
references.pmid: 17952055
references.citation: Biochemistry. 2002 Nov 19;41(46):13681-9
references.pmid: 12427030
references.citation: Curr Opin Cell Biol. 1997 Apr;9(2):134-42
references.pmid: 9069253
references.citation: Annu Rev Biochem. 2001;70:703-54
references.pmid: 11395421
references.citation: Biochem Biophys Res Commun. 2006 Jan 13;339(2):695-700
references.pmid: 16325780
references.citation: Semin Cell Dev Biol. 1998 Apr;9(2):129-34
references.pmid: 9599407
references.citation: Science. 2000 Aug 4;289(5480):739-45
references.pmid: 10926528
references.citation: Science. 2007 Nov 23;318(5854):1258-65
references.pmid: 17962520
references.citation: Mol Cell Proteomics. 2005 Mar;4(3):310-27
references.pmid: 15665377
references.citation: Recent Prog Horm Res. 1996;51:319-51; discussion 352-3
references.pmid: 8701085
references.citation: J Pept Res. 1998 Aug;52(2):95-106
references.pmid: 9727865
references.citation: Mol Cell Biol. 1998 Oct;18(10):5981-91
references.pmid: 9742115
references.citation: Nat Biotechnol. 2002 Mar;20(3):301-5
references.pmid: 11875433
references.citation: Mol Biol Cell. 2009 Dec;20(24):5039-50
references.pmid: 19828733
references.citation: Sci STKE. 2006 Dec 05;2006(364):cm6
references.pmid: 17148787
references.citation: J Biol Chem. 1988 Mar 15;263(8):3893-7
references.pmid: 2831211
references.citation: Genetics. 2006 Mar;172(3):1459-76
references.pmid: 16361226
references.citation: Cell. 1992 Nov 27;71(5):755-63
references.pmid: 1330324
references.citation: Cell. 2006 Sep 22;126(6):1079-93
references.pmid: 16990133
references.citation: Biochemistry. 2001 Jan 30;40(4):1102-8
references.pmid: 11170434
references.citation: Mol Cell Biol. 2000 Jul;20(14):5350-9
references.pmid: 10866691
references.citation: Methods Enzymol. 1987;154:367-82
references.pmid: 3323813