Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization

Abstract

G protein-coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The alpha-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding alpha-factor. Ubiquitination triggers Ste2p internalization into the endocytic pathway. Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization. Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis. The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence. Mutants defective in yeast casein kinase I homologues are unable to internalize alpha-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.

Figure 1

Sequences of mutant Ste2p cytoplasmic tails.

Figure 2

Ubiquitination mediates constitutive receptor internalization. (A) The SINNDAKSS serines and lysine are required for internalization of the 345Stop truncated receptor in the absence of α-factor. Strains expressing different forms of the 345Stop receptor (RH3180, RH3181, and RH3333) were treated with cycloheximide to inhibit new receptor synthesis and then harvested in the presence of energy poisons at various times. The relative number of receptors remaining at the plasma membrane at each time was determined by incubating cells with radiolabeled α-factor to measure the extent of cell surface pheromone binding. This value is expressed as the amount of α-factor bound at each time point relative to that bound at t = 0 min. This experiment is representative of three independent experiments performed on these strains. 345Stop, (closed diamonds); K337R, 345Stop, (open circles); 3S→ A, 345Stop (open squares). (B) The α-factor receptor is ubiquitinated in the absence of pheromone. Cell extracts were prepared from wild-type (RH448), end4Δ (RH1965), and ste2Δ (RH1298) strains that had never been exposed to α-factor. Immunoblots were prepared from these extracts and then probed with anti-Ste2p antiserum. Ubiquitinated and phosphorylated forms of the receptor are indicated by brackets (Hicke and Riezman, 1996; Terrell et al., 1998).

Figure 2

Ubiquitination mediates constitutive receptor internalization. (A) The SINNDAKSS serines and lysine are required for internalization of the 345Stop truncated receptor in the absence of α-factor. Strains expressing different forms of the 345Stop receptor (RH3180, RH3181, and RH3333) were treated with cycloheximide to inhibit new receptor synthesis and then harvested in the presence of energy poisons at various times. The relative number of receptors remaining at the plasma membrane at each time was determined by incubating cells with radiolabeled α-factor to measure the extent of cell surface pheromone binding. This value is expressed as the amount of α-factor bound at each time point relative to that bound at t = 0 min. This experiment is representative of three independent experiments performed on these strains. 345Stop, (closed diamonds); K337R, 345Stop, (open circles); 3S→ A, 345Stop (open squares). (B) The α-factor receptor is ubiquitinated in the absence of pheromone. Cell extracts were prepared from wild-type (RH448), end4Δ (RH1965), and ste2Δ (RH1298) strains that had never been exposed to α-factor. Immunoblots were prepared from these extracts and then probed with anti-Ste2p antiserum. Ubiquitinated and phosphorylated forms of the receptor are indicated by brackets (Hicke and Riezman, 1996; Terrell et al., 1998).

Figure 3

The SINNDAKSS serines are required for receptor phosphorylation. Incubation of mutant 345Stop receptors with protein phosphatase. Cells expressing different variants of the 345Stop α-factor receptor were radiolabeled with EXPRE³⁵S³⁵S protein–labeling mix and then lysed before (−) or 10 min after (+) exposure to α-factor. The receptors were then isolated by immuneprecipitation, incubated with phosphatase (+) or subjected to a mock incubation (−), resolved by SDS-PAGE, and then detected by autoradiography. The experiment shown here was performed with protein phosphatase 1 that is specific for serine/threonine phosphorylation; however, identical results were obtained with alkaline phosphatase.

Figure 4

Mutation of the SINNDAKSS serines diminishes both constitutive and stimulated receptor internalization rates in the context of the full-length Ste2p tail. (A) The clearance of wild-type (RH448, closed circles) and 3S→ A (RH3687, open circles) receptors from the cell surface in the absence of α-factor.(B) The clearance of wild-type (closed circles) and 3S→ A (open circles) receptors from the cell surface in the presence of α-factor. (C) α-Factor ligand internalization assays performed on cells expressing either wild-type (closed circles) or 3S→ A receptors (open circles). All curves represent the average of three independent experiments.

Figure 5

The substitution of negatively charged amino acids for the SINNDAKSS serines rescues ligand-stimulated but not constitutive receptor internalization. (A) α-Factor internalization assays performed on strains expressing 345Stop (closed diamonds), 3S→ A, 345Stop (open triangles), 3S→ E, 345Stop (open squares), or 3S→ D, 345Stop (open circles) receptors. These curves represent the averages of at least two independent experiments. (B) Receptor clearance assays performed in the absence of pheromone on strains expressing 345Stop (closed diamonds) or 3S→ E, 345Stop (open squares) receptors. These curves are representative of three independent experiments.

Figure 5

The substitution of negatively charged amino acids for the SINNDAKSS serines rescues ligand-stimulated but not constitutive receptor internalization. (A) α-Factor internalization assays performed on strains expressing 345Stop (closed diamonds), 3S→ A, 345Stop (open triangles), 3S→ E, 345Stop (open squares), or 3S→ D, 345Stop (open circles) receptors. These curves represent the averages of at least two independent experiments. (B) Receptor clearance assays performed in the absence of pheromone on strains expressing 345Stop (closed diamonds) or 3S→ E, 345Stop (open squares) receptors. These curves are representative of three independent experiments.

Figure 6

The substitution of negatively charged amino acids for the SINNDAKSS serines rescues ligand-stimulated receptor phosphorylation. (A) Cells expressing 345Stop (RH3180) or 3S→ E, 345Stop (RH3510) receptors were radiolabeled with EXPRE³⁵S³⁵S protein–labeling mix and then lysed before (−) or 10 min after (+) incubation with α-factor. Radiolabeled receptors were isolated from cell extracts by immuneprecipitation. The immuneprecipitates were then incubated with alkaline phosphatase (+) or subjected to a mock incubation (−), resolved by SDS-PAGE, and then receptors were detected by autoradiography. (B) Cultures of cells expressing 345Stop (LHY638), 3S→ A, 345Stop (LHY639), or 3S→ E, 345Stop (LHY636) receptors were propagated and then split into two identical aliquots. One aliquot was metabolically radiolabeled with H₃ ³²PO₄; the other aliquot was radiolabeled with Tran³⁵SLabel protein–labeling mix. Radiolabeled cells were lysed before (−) or 10 min after (+) incubation with α-factor and then receptors were immuneprecipitated and resolved by SDS-PAGE. The mobility difference between phosphorylated and nonphosphorylated forms of the receptor is not as pronounced as observed in Fig. 6 A because a different percentage polyacrylamide gel was used to resolve these receptors. (C) The amount of radioactive receptor detected in each immuneprecipitation was determined by analysis with ImageQuant software (Molecular Dynamics). The level of phosphorylated receptor precipitated was normalized to the amount of ³⁵S-labeled receptor precipitated from each strain to correct for differences in the expression level of different receptors. The relative level of phosphorylation in each sample is expressed as a ratio of the phosphorylation of the 345Stop receptor in the absence of α-factor.

Figure 7

Mutants carrying defective Yck kinases are unable to modify or internalize α-factor receptor. (A) α-Factor internalization assays performed on wild-type (RH3992), end4 (RH1965), and yck1Δ yck2-2 (RH3589) strains preincubated for 15 min at 24° or 37°C. Wild-type, 24°C, closed circles; wild-type, 37°C, open circles; yck1Δ yck2-2ρ, 24°C, closed diamonds; yck1Δ yck2-2, 37°C, open diamonds; end4, 37°C, open squares. (B) Immunoblot of extracts prepared from yck1Δ yck2-2 (RH3589) and end4 (RH1597) cells before (−) or 10 min after (+) exposure to α-factor. Each strain was preincubated for 15 min at 37°C. Cells were removed for the no α-factor sample and then incubation was continued at 37°C after the addition of α-factor. Extracts were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with α-Ste2p antiserum. Modified forms of the receptor are designated by the bracket. (C) Immuneprecipitates of Ste2p from end4 and yck1Δ yck2-2 strains incubated at 37°C. Cells were labeled with EXPRE³⁵S³⁵S protein–labeling mix for 30 min at 30°C, chase mix was added, and then the cells were shifted to 37°C for 15 min before withdrawing samples before (−) and 10 min after (+) exposure to α-factor. Cell extracts were prepared and precipitated with Ste2p antiserum. The precipitates were incubated with protein phosphatase 1 (+) or mock-treated with buffer alone (−) and then resolved by SDS-PAGE. The precipitated proteins were visualized by autoradiography.

Figure 8

Cells expressing receptors that are internalized inefficiently are severely defective in their ability to recover from cell cycle arrest induced by α-factor binding. Cells expressing 345Stop (closed diamonds), K337R, 345Stop (open circles), 3S→ A, 345Stop (open triangles), or 3S→ E, 345Stop (open squares) receptors were incubated with α-factor until the entire population lacked buds, indicating a full arrest of growth in the G1 phase of the cell cycle. α-Factor was then washed away from the cells and the number of budded cells in each culture was counted after various periods of time as a measure of the ability of cells to recover from growth arrest and resume growing. Each recovery curve is an average of at least three independent experiments. The standard deviation at each time point is indicated by error bars.