Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction

Zanolari, Bettina; Raths, Susan; Singer-Krüger, Birgit; Riezman, Howard

doi:10.1016/0092-8674(92)90552-n

Cited by 71 publications

(63 citation statements)

References 44 publications

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“…However, one may not conclude that internalization is needed for the trophic effect to occur, since CQ, MA, and SW prevent binding. Signal transduction has been shown to be independent of receptor-ligand endocytosis in several instances (44)(45)(46) and dependent on endocytosis in some (47), but our data do not allow us to discriminate between the two possibilities.…”

Section: Resultscontrasting

confidence: 62%

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

Lew

Songu-Mize²,

Pontow³

et al. 1994

J. Clin. Invest.

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IntroductionThe putative mannose receptor (MR), previously implicated in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle (ASM) cells, was studied to determine its properties. (7). The mitogenic effect is blocked by yeast mannan, a mannose receptor (MR) blocker (8), but not by mannose-6-phosphate, a specific blocker of the cation independent mannose-6-phosphate/insulin-like growth factor II receptor (9), suggesting the involvement of a mannose-recognizing moiety (7).The well-studied mannose receptor of macrophages (M0MR) is involved in receptor-mediated endocytosis and molecular scavenging of ManGP as well as phagocytosis and host defense against mannosyl-rich pathogens (10-12). Mannosylrich enzymes such as lysosomal hydrolases including Hex (11), myeloperoxidase (13), and plasminogen activator (14)

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Section: Resultscontrasting

confidence: 62%

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

Lew

Songu-Mize²,

Pontow³

et al. 1994

J. Clin. Invest.

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“…Because this finding indicates that the third cytoplasmic loop can provide a signal for receptor internalization, ligand-dependent endocytosis of wildtype receptors may also involve the third cytoplasmic loop. Pheromone binding may induce a conformational change in the third loop which is transmitted to the C-terminal cytoplasmic domain of the ac-factor receptor, a region known to be required for receptor endocytosis (38,41,51). Although this model is similar to a previous one (51) in which the ligandbound (and presumably active) conformation of the receptor triggers endocytosis, it is distinct in the following way.…”

Section: Discussionmentioning

confidence: 90%

“…Pheromone binding may induce a conformational change in the third loop which is transmitted to the C-terminal cytoplasmic domain of the ac-factor receptor, a region known to be required for receptor endocytosis (38,41,51). Although this model is similar to a previous one (51) in which the ligandbound (and presumably active) conformation of the receptor triggers endocytosis, it is distinct in the following way. Because we find that mutant receptors undergoing constitutive endocytosis do not constitutively activate the pheromone response pathway, we suggest that the third cytoplasmic loop can adopt different conformations for signalling endocytosis and Gprotein activation.…”

Section: Discussionmentioning

confidence: 90%

The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.

Stefan¹,

Blumer²

1994

Mol. Cell. Biol.

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To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the a-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2's alleles were identified by genetic screening methods that increased oa-factor sensitivity 2.5-to 15-fold. The phenotypic effects of ste2'ss and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the a-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2"sf mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an a-factor antagonist. One ste2"sf allele increased receptor binding affinity for a-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonistresponsive phenotypes caused by ste2' alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2s' mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the a-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.Cell surface receptors that are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) function in signal transduction pathways that allow eukaryotic cells to respond to various hormones, neuroregulatory molecules, and sensory stimuli. In these pathways, receptors program cellular responses by interacting specifically with certain ligands and G proteins. G-protein-coupled receptors are subjected to regulatory processes involving receptor phosphorylation, sequestration, and down-regulation which limit the strength or duration of physiological responses (4,15,36).Recent investigations are beginning to address the molecular mechanisms by which receptors are activated by specific ligands and undergo agonist-induced sequestration and downregulation. An emerging theme is that membrane-spanning as well as cytoplasmic domains of G-protein-coupled receptors can have roles in these processes (12,18,22,39,40,48). Mutagenesis and domain swapping experiments have indicated, for example, that the third cytoplasmic loops of certain mammalian receptors control G-protein activation, receptor sequestration, and responses to partial agonists (6, 10, 11, 13, 19, 28-30, 42, 43). Indeed, in ,-adrenergic receptors, discrete subdomains of the third cytoplasmic loop apparently control subsets of these processes, because mutant...

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“…Furthermore, an intact signal transduction pathway is not required for receptor endocytosis. Mutations in the STE4 gene, encoding the a subunit of the heterotrimeric G protein, or in the STE5 gene block signal transduction events but have no effect on the down regulation of the receptor (30,63), whereas mutations in the GPAJ gene, encoding the G protein a subunit, cause constitutive activation of pheromone response without affecting the ability of receptors to undergo down regulation (4). Pheromone receptors expressed from a plasmid under the control of the GAL promoter in diploid cells were competent for endocytosis, even though diploid cells do not have a functional pheromone response pathway (19,63).…”

Section: Discussionmentioning

confidence: 99%

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

Schandel¹,

Jenness²

1994

Mol. Cell. Biol.

132

173

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When Saccharomyces cerevisiae a cells bind a-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for a-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the a-factor receptors were predominantly found in the plasma membrane peak before a-factor treatment and then appeared in membranes of lesser buoyant density after a-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before a-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. presumably by interacting with a common G protein (6, 29), they share no obvious sequence homology. Mutational studies of the a-factor receptor (17, 61) and other receptors with a similar structure (23, 32) implicate the third cytoplasmic loop in the coupling of these receptors to their respective G proteins. Other mutational analyses indicate that the carboxyterminal cytoplasmic domain of the pheromone receptor is important for receptor down regulation, pheromone internalization, adaptation to the pheromone-induced signal, and pheromone-induced morphological changes (33,34,47,49).A large body of experimental evidence suggests that pheromones are internalized by receptor-mediated endocytosis. Radioactive at-factor becomes associated irreversibly with a cells in an energy-and receptor-dependent manner (14,30). This association is accompanied by a disappearance of ligandbinding sites from the cell surface (30). Clathrin, which plays a major role in receptor-mediated endocytosis in mammalian cells, has recently been shown to facilitate the internalization of a-factor (58). Following internalization, the pheromone appears to be translocated to the vacuole via vesicular intermediates (54), which may represent early and late endosomes (55). Although there is no direct evidence that the ao-factor receptor leaves the cell surface upon binding pheromone, a-factor receptors have been shown to pass from the plasma membrane to the vacuole, and this process is accelerated when at cells are treated with a-factor (19). There is as yet no direct evidence for the internalization of the a-factor ligand. We sought physical evidence for the ligand-induced endocytosis of the a-factor receptor. Three independent criteria were used to establish the movement of a-factor receptors from the plasma membrane to internal compartments of the cell. First, Renografin density gradient centrifugation was used to fractionate the cellular membranes, allowing us to follow the ligand-induced exit of the receptor from the plasma membrane upon at-factor exposure. Second, protease susceptibility assays 7245

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Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction

Cited by 71 publications

References 44 publications

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

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