Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway.

Weiner, Jennifer L.; Guttierez-Steil, C; Blumer, Kendall J.

doi:10.1016/s0021-9258(18)53064-7

Cited by 55 publications

(15 citation statements)

References 45 publications

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“…The current model of signal transduction by G proteincoupled receptors involves the isomerization of the receptor from an inactive to an active form. Using mutational analysis, the sixth transmembrane domain and the third cytoplasmic loop were implicated in receptor activation (51)(52)(53) and polar residues in transmembrane domain 6 of Ste2p were suggested to interact with other domains of the receptor (54). Previously we reported that an 18-residue homologue, M6-18L (252-269, C252A, P258L) corresponding to the constitutively active receptor Ste2p-P258L, was a highly R-helical structure in SDS micelles but was a β-structure in HFA-water (20).…”

Section: Discussionmentioning

confidence: 99%

Synthesis and Biophysical Analysis of Transmembrane Domains of a Saccharomyces cerevisiae G Protein-Coupled Receptor

et al. 2000

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The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.

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Section: Discussionmentioning

confidence: 99%

Synthesis and Biophysical Analysis of Transmembrane Domains of a Saccharomyces cerevisiae G Protein-Coupled Receptor

et al. 2000

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“…because a dominant-interfering phenotype (attenuation of agonist-induced growth arrest and/or reporter gene expression) was still observed when: the ability of the mutant receptor to interact with G proteins was disrupted (by introducing the L236R substitution in cis in its third cytoplasmic loop which we previously showed does not affect agonist binding affinity or receptor expression [13]; Figure 3a,d,g,j); G protein α, β and γ subunits were overexpressed approximately 10-20-fold from a high-copy plasmid carrying the genes encoding each subunit expressed from their normal promoters (Figure 3e-g); and the wild-type receptor was fused to the G α subunit (Figure 3h-j). Third, overexpression of mutant receptors (10-fold from the PGK1 promoter) blocked FRET between CFP-and YFP-tagged tailless wild-type receptors (data not shown) without affecting expression of tagged receptors, as indicated by quantitation of CFP and YFP fluorescence and fluorescence microscopy.…”

Section: Figurementioning

confidence: 99%

G-protein-coupled receptors function as oligomers in vivo

2000

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Hormones, sensory stimuli, neurotransmitters and chemokines signal by activating G-protein-coupled receptors (GPCRs) [1]. Although GPCRs are thought to function as monomers, they can form SDS-resistant dimers, and coexpression of two non-functional or related GPCRs can result in rescue of activity or modification of function [2-10]. Furthermore, dimerization of peptides corresponding to the third cytoplasmic loops of GPCRs increases their potency as activators of G proteins in vitro [11], and peptide inhibitors of dimerization diminish beta(2)-adrenergic receptor signaling [3]. Nevertheless, it is not known whether GPCRs exist as monomers or oligomers in intact cells and membranes, whether agonist binding regulates monomer-oligomer equilibrium, or whether oligomerization governs GPCR function. Here, we report that the alpha-factor receptor, a GPCR that is the product of the STE2 gene in the yeast Saccharomyces cerevisiae, is oligomeric in intact cells and membranes. Coexpression of receptors tagged with the cyan or yellow fluorescent proteins (CFP or YFP) resulted in efficient fluorescence resonance energy transfer (FRET) due to stable association rather than collisional interaction. Monomer-oligomer equilibrium was unaffected by binding of agonist, antagonist, or G protein heterotrimers. Oligomerization was further demonstrated by rescuing endocytosis-defective receptors with coexpressed wild-type receptors. Dominant-interfering receptor mutants inhibited signaling by interacting with wild-type receptors rather than by sequestering G protein heterotrimers. We suggest that oligomerization is likely to govern GPCR signaling and regulation.

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“…To create the STE2-KEX2 chimera, PCR fragments encoding relevant portions of STE2 and KEX2 were produced with the creation of a unique BglII site at the chimera junction. A 750-bp fragment of STE2 (Nakayama et al, 1985) encoding amino acids 45-297 was amplified from pRS314-STE2 (Weiner et aL, 1993) using the primers 5'-GCTTCTAGAGTTAA-CAGTACTGTTACTCAG-Y (primer A) and 5'-GGAAGATCTCGTG-GCCCACATTGATGA-3'. A 650-bp fragment of KEX2 (Fuller et al, 1989) that encodes the entire cytoplasmic tail of Kex2p from amino acids 701-814 as well as roughly 300 bases of the 3' untranslated region was amplified from pJ2B (Julius et aL, 1984) with the primers 5'-GGAAGATCT-TCAAGGAGAAGGATCAGA-3' (primer B) and 5'-CGCGGATC-CTIT/'TAATACACCAAAGA-3' (primer C).…”

Section: Construction Of Stex22 and Mutant Derivativesmentioning

confidence: 99%

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae.

Tan¹,

Howard²,

Payne³

1996

The Journal of Cell Biology

101

103

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Abstract. The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the a-factor receptor, Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a-factor receptor, Ste3p. Mutation of this sequence prevented pheromone-stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysinebased, ubiquitin-dependent endocytosis signals in yeast. C LATHRIN-mediated endocytosis of plasma membranereceptors promotes the rapid and efficient uptake of receptor-bound ligands, typically nutrients and signaling molecules important for cell growth and differentiation. Plasma membrane proteins subject to efficient endocytosis contain specific, cytoplasmically disposed amino acid sequences that are necessary for uptake (for review see Trowbridge et al., 1993). Such endocytic targeting signals often contain an aromatic amino acid (tyrosine or phenylalanine) and serve to direct proteins into clathrincoated pits. The critical importance of aromatic amino acids in the targeting sequences has been established by mutational studies (for review see Trowbridge et al., 1993). For example, mutation of tyrosine 807 in the low-density lipoprotein (LDL) 1 receptor disrupts clathrin-coated pit localization and thereby prevents uptake from the plasma membrane (Davis et al., 1986(Davis et al., , 1987 the influenza hemagglutinin protein dramatically increases the efficiency of internalization via clathfin-coated pits (Lazarovits and Roth, 1988). In addition to the aromatic amino acid--containing motifs, there are also endocytic targeting signals that lack aromatic amino acids but appear to mediate internalization through clathrin-coated pits. Examples of these signals include di-leucine motifs in the CD3 subunits of the T cell antigen receptor (Letourneur and Klausner, 1992) and a lysine-containing signal in the yeast a-factor receptor, Ste2p (Rohrer et al., 1993).The location of endocytic t...

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Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway.

Cited by 55 publications

References 45 publications

Synthesis and Biophysical Analysis of Transmembrane Domains of a Saccharomyces cerevisiae G Protein-Coupled Receptor

Synthesis and Biophysical Analysis of Transmembrane Domains of a Saccharomyces cerevisiae G Protein-Coupled Receptor

G-protein-coupled receptors function as oligomers in vivo

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae.

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