Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast.

Rohrer, Jack; Bénédetti, Hélène; Zanolari, Bettina; Riezman, Howard

doi:10.1091/mbc.4.5.511

Cited by 123 publications

(141 citation statements)

References 42 publications

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“…This value is in rough agreement with, but slightly faster than, the rate of receptor endocytosis (t 0.5 ϭ Ϸ4.5 min) measured by labeled ␣-factor internalization (13). To further investigate this issue, the wild-type ␣-factor receptor was replaced with a mutant version lacking the C-terminal tail beyond residue 300 so that ligandstimulated endocytosis was severely impaired (14). G protein activation was not detrimentally affected, but G protein deactivation was dramatically curtailed in the mutant (Fig.…”

Section: Resultssupporting

confidence: 65%

“…The measured K d for ␣-factor binding to truncated receptor was elevated to 15 nM, representing a decrease in receptor affinity. Because tail-less Ste2p is endocytosed at a slower rate (14), the steady-state numbers of receptors on the cell surface were increased from Ϸ10,000 molecules per cell to Ϸ60,000 molecules per cell. Raising the number of surface receptors may partially explain the more sensitive response.…”

Section: Dose-response Curves To ␣-Factor In Wild-type and Mutant Cellsmentioning

confidence: 99%

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A quantitative characterization of the yeast heterotrimeric G protein cycle

Kitano

2003

Proc. Natl. Acad. Sci. U.S.A.

171

188

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The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-G␣ and G␤␥-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose-response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of ␣-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose-response curve and the downstream dose-response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. Living organisms detect and respond to a variety of environmental cues through heterotrimeric G protein signal transduction pathways. Genetic or pharmacologic perturbation of these systems in humans can have significant medical consequences (1). Many pharmaceutical agents are directed against components of the G protein activation͞deactivation cycle (1). Achieving a quantitative understanding of this cycle and of the relationship between G protein activation and downstream physiology can aid in drug development.One of the best studied heterotrimeric G protein signaling systems is the yeast mating response in Saccharomyces cerevisiae (2, 3). Haploid yeast cells respond to a peptide pheromone from their partner by undergoing a series of events (e.g., cell-cycle arrest, synthesis of new proteins) in preparation for mating. In a cells, the pheromone ␣-factor, secreted by ␣ cells, activates the G protein coupled ␣-factor receptor (Ste2p). Most of the components of this pathway have been identified, including the receptors Ste2p and Ste3p (a-factor receptor), the G protein subunits Gpa1p (G␣), Ste4p (G␤), and Ste18p (G␥), and the G protein regulator (RGS) Sst2p.Yeast geneticists have isolated mutants possessing altered sensitivity to the pheromone ␣-factor (4). In particular, the following all confer an ␣-factor supersensitive phenotype: the deletion of BAR1 (bar1⌬), which encodes a secreted protease that degrades ␣-factor, the deletion of SST2 (sst2⌬), and the removal of the C-terminal tail of ␣-factor receptor STE2 (ste2 300⌬). It is important to provide a quantitative description of how these mutations affect the G protein cycle to complement the existing qualitative understanding.Until recently, the ...

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Section: Resultssupporting

confidence: 65%

Section: Dose-response Curves To ␣-Factor In Wild-type and Mutant Cellsmentioning

confidence: 99%

A quantitative characterization of the yeast heterotrimeric G protein cycle

Kitano

2003

Proc. Natl. Acad. Sci. U.S.A.

171

188

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“…Agonist binding induces internalization of Ste2p by a clathrin-dependent mechanism, and the internalized receptor is transported to the vacuole (the yeast equivalent of a lysosome), where it is degraded [223,224]. The sequence SINN-DAKSS within the C-terminal tail of Ste2p is necessary and sufficient for receptor endocytosis [225]. Ligand binding induces ubiquitination of the Ste2p C-terminal tail, which is required for endocytosis, since internalization is 5-15-fold slower in yeast mutants that lack multiple ubiquitin-conjugating enzymes [222].…”

Section: Ubiquitin-mediated Proteolysismentioning

confidence: 99%

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

Böhm

Grady

Bunnett

1997

Biochemical Journal

477

322

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The large and functionally diverse group of G-protein-coupled receptors includes receptors for many different signalling molecules, including peptide and non-peptide hormones and neurotransmitters, chemokines, prostanoids and proteinases. Their principal function is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins, and they thereby participate in many aspects of regulation. Cellular responses to agonists of these receptors are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis and downregulation. Agonists are removed by dilution, uptake by transporters and enzymic degradation. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second-messenger kinases, interaction of phosphorylated receptors with arrestins and receptor uncoupling from

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“…Nonetheless, as with the clathrin-mediated uptake, the selectivity of the process ultimately depends upon the recognition of determinants or signals encoded within the endocytic substrate's amino acid sequence. Sequences involved in directing uptake have been identified for several yeast endocytic substrates (1,(12)(13)(14)(15). Given the differences between clathrin-and ubiquitindependent uptake processes, it is not surprising that these yeast sequences bear little resemblance to the signals directing the clathrin-mediated endocytosis of mammalian cells.…”

mentioning

confidence: 99%

“…constitutive) uptake mechanism. For Ste2p, the sequence SINNDAKSS, which maps to the regulatory C-terminal cytoplasmic tail do-main (CTD), 1 has been shown to be required for ␣-factor-induced uptake (12,16). Mutations in this sequence block both ubiquitination and endocytosis.…”

mentioning

confidence: 99%

Ubiquitination of the PEST-like Endocytosis Signal of the Yeast a-Factor Receptor

Roth¹,

Davis²

2000

Journal of Biological Chemistry

104

110

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A 58-residue-long, PEST-like sequence within the yeast a-factor receptor (Ste3p) specifies the ubiquitination, endocytosis, and consequent vacuolar degradation of the receptor protein (Roth, A. F., Sullivan, D. M., and Davis, N. G. (1998) J. Cell Biol. 142, 949 -961). The present work investigates three lysyl residues that map within this sequence as the potential ubiquitin acceptor sites. Lys 3 Arg substitution mutants were tested for effects on both ubiquitination and endocytosis. Results indicate that the three lysines function redundantly; a severe blockade to both ubiquitination and endocytosis is seen only for receptors having all three lysines replaced. Of the three, Lys 432 plays the predominant role; ubiquitination and turnover are significantly impaired for receptors having just the K432R mutation. CNBr fragmentation of the receptor protein, used for the physical mapping of the ubiquitin attachment sites, showed PEST-like sequence lysines to be modified both with single ubiquitin moieties as well with short multi-ubiquitin chains, two or three ubiquitins long. Thus, in addition to being the signal for ubiquitination, the Ste3p PEST-like sequence also provides the site for ubiquitin attachment. To test if this endocytosis signal functions solely for ubiquitination, we have asked if the requirement for the PEST-like sequence in endocytosis might be bypassed through pre-attachment of ubiquitin to the receptor protein. Indeed, Ste3-ubiquitin translational fusions that have a ubiquitin moiety fused to the receptor in place of the PEST-like signal do undergo rapid endocytosis and vacuolar turnover. We conclude that ubiquitin alone, with no required contribution from receptor sequences, provides the sufficient signal for initiating uptake. In addition, our results confirm conclusions originally drawn from studies with the ␣-factor receptor (Terrell, J., Shih, S., Dunn, R., and Hicke, L. (1998) Mol. Cell 1, 193-202), namely that mono-ubiquitin, and not multi-ubiquitin chains provide the primary recognition determinant for uptake. Although mono-ubiquitination suffices, our results indicate that multi-ubiquitination serves to augment the rate of uptake.

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Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast.

Cited by 123 publications

References 42 publications

A quantitative characterization of the yeast heterotrimeric G protein cycle

A quantitative characterization of the yeast heterotrimeric G protein cycle

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

Ubiquitination of the PEST-like Endocytosis Signal of the Yeast a-Factor Receptor

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