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

Cell, Vol. 71, 755-763,

November

27, 1992, Copyright

0 1992 by Cell Press

Yeast Pheromone Receptor Endocytosis and Hyperphosphorylation Are Independent of G Protein-Mediated Signal Transduction Bettina Zanolari,’ Susan Raths,‘t Birgit Singer-Kruger,** and Howard l Biocenter of the University of Base1 Klingelbergstrasse CH-4056 Base1 Switzerland

Riezman’

70

Summary When a factor binds to the yeast a factor receptor a signal is transmitted via a tripartite G protein that prepares the cell for conjugation. As a result of a factor binding the receptor also undergoes a regulated internalization and hyperphosphorylation. Using cells that lack activity of this tripartite G protein, we show that G protein-mediated pheromone signal transduction is not necessary for regulation of receptor internalization or hyperphosphorylation. Therefore, the processes of signal transduction and down regulation can be uncoupled. We propose that binding of a factor to its receptor results in a receptor conformation change that permits receptor hyperphosphorylation and interaction with the endocytic machinery. Introduction Saccharomyces cerevisiae cells exist in two haploid cell types, a and a, that can mate to form the a/a diploid cell. The conjugation pathway is initiated by the binding of secreted peptide mating pheromones, called a and a factor, to receptors on the surface of cells of the opposite mating type. This binding initiates a signal transduction cascade that is amplified by the action of a haploid cell-specific tripartite G protein (Dietzel and Kurjan, 1987; Miyajima et al., 1987; Whiteway et al., 1989; Blinder et al., 1989). Signal transduction leads to an arrest of the cell cycle in Gl, the development of a pear-shape cell morphology, called a “shmoo,” and changes in gene expression (for review see Cross et al., 1988; Marsh et al., 1991). Some of these changes in gene expression are likely to be mediated by phosphorylation of Stel2p, a transcription factor (Errede and Ammerer, 1989; Dolan et al., 1989). This may occur via a kinase cascade involving the putative protein kinases Ste7p and Stel 1 p that work upstream of Stel2p and are essential for mating (see Fields, 1990). The receptors for the mating pheromones are polytopic membrane proteins (Nakayama et al., 1985; Burkholder and Hartwell, 1985; Hagen et al., 1986) with a structure similar to other G protein coupled receptors such as rhodopsin and the f3-adrenergic receptor (Dixon et al., 1986). The a factor receptor is encoded by the STE2 gene (Jen-

tPresent address: Department of Biology, Universtty of Mississippi, Oxford, Mississippi. *Present address: European Molecular Biology Laboratory, Cell BIoIogy Program, Heidelberg, 6900 Germany.

ness et al., 1983). Upon a factor binding to the receptor, the ligand is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko et al., 1986; Jenness and Spatrick, 1986) while there is a simultaneous disappearance of cell-surface a factor binding activity, indicating down regulation (Jenness and Spatrick, 1986). The a factor is then degraded (Chvatchko et al., 1986) after transport via vesicular intermediates to the vacuole (Singer and Riezman, 1990). It is likely that the receptor is also degraded, as it does not seem to recycle back to the cell surface (Jenness and Spatrick, 1986). Upon binding a factor, the receptor is hyperphosphorylated on its cytoplasmic tail (Reneke et al., 1988). This phosphorylation has been postulated to play a role in recovery from pheromone action (Reneke et al., 1988; Konopka et al., 1988) as deletion of the cytoplasmic tail results in a defect in the adaptation process. However this deletion also caused a defect in receptor-mediated endocytosis (Reneke et al., 1988). To understand how the internalization of a factor and its receptor is regulated, we have expressed the receptor in cells deficient for pheromone-specific G protein-mediated signal transduction. We show that proper regulation of receptor-mediated endocytosis and hyperphosphorylation occurred in the absence of G protein-mediated signal transduction. Our results suggest that binding of a factor to the receptor induces it to undergo a conformational change that allows it to be endocytosed and hyperphosphorylated. Therefore, receptor down regulation and hyperphosphorylation are not induced as a feedback control that is regulated by the activation of the mating, protein kinase cascade.

Results To test receptor down regulation and hyperphosphorylation in the absence of G protein-mediated signal transduction, we constructed two systems. In one, the ST& gene, encoding the 6 subunit of the haploid-specific tripartite G protein, wasdisrupted in a haploid MATa cell. This renders the cells sterile as tested by their inability to mate (Whiteway et al., 1989). In the other system, we introduced a plasmid (plHl-9) encoding the STE2 gene under galactose control (see Experimental Procedures) into diploid cells. Diploid cells lack pheromone receptors, the tripartite G protein involved in mating, and several other haploidspecific gene products (Herskowitz, 1988,1989; Sprague, 1990). To verify that diploid cells expressing Ste2p did not respond to pheromone, we evaluated their response to a factor by morphological observations. As a control we used haploid cells that contained a deletion in the chromosomal STE2 gene and carried the plasmid plHl-9. After a 5.5 hr incubation with a factor at 30°C, the haploid cells grown on galactose as a carbon source (thus expressing receptors) responded to 1 O-” M a factor, whereas the same cells grown with glucose as a carbon source (not expressing receptors) did not respond to a lo-fold higher concen-

Cell 756

Figure 1. a/u Diploid Cells Expressing Do Not Respond to CI Factor

STE2

Strain RH1199 (haplord, Aste2) transformed with plasmid plHl-9 was grown overnight on SD-ura (glucose, panel A) or SGal-ura (galactose, panel B) medrum. Strain RH765 transformed with plHl-9 (a/a diploid, panels C and D) was grown overnight on SGal-ura medium. The cells were treated at 30°C for 5.5 hr with 10 ‘Ma factor (panel B), 10 ‘M a factor(panels A and D), or no a factor (panel C). The cells were vrsualized by Nomarsky optics usrng a 63x objective on the Zeiss Axrophot microscope. Magnificatron = 850 x

tration of pheromone (Figure 1). Diploid cells grown on galactose also did not respond to 10m5 M a factor. Receptor Internalization Is Independent of G Protein-Mediated Signal Transduction To test the ability of nonresponsive cells to internalize a factor, we grew wild-type and ste4 disrupted cells overnight in YPUAD (1% yeast extract, 2% peptone, 40 pg/ml uracil, 40 kg/ml adenine sulfate, 2% glucose) medium. The log phase cells were washed, and radioactive a factor was bound at 0°C. The cells were then washed, warmed to 24% for various times, and washed again at neutral or acid pH. The acid pH wash removes surface-bound pheromone, allowing the quantitation of internalized a factor. The ste4 mutant cells bound approximately 50% less ligand than the isogenic wild type (34% of total a factor bound versus 17%) consistent with previous results (Jenness et al., 1983) and consistent with the lowered affinity of the receptor for a factor when the G protein is nonfunctional (Blumer and Thorner, 1990). However, as demonstrated in Figure 2, the kinetics of internalization of the bound a factor were identical for wild-type and ste4 mutant cells. These results show that ligand internalization is independent of G protein-mediated signal transduction. Once the ligand is bound it is cleared from the cell surface with normal kinetics. To test whether the other components of the tripartite G protein could be involved in receptor internalization, we tested uptake in receptor-bearing diploid cells as described above. a factor uptake by diploid cells is indistinguishable from uptake by haploid cells (Figure 2). There-

fore, none of the three subunits of the tripartite G protein play a role in ligand internalization. The G protein could also play a role in regulating endocytosis. By binding to the receptor, the G protein could retain the receptor at the surface until a ligand-dependent dissociation occurs. If this were the case, then receptor internalization should be constitutive in cells lacking the G protein. We tested for surface receptor retention and ligandinduced receptor clearance in responding and nonresponding cells. Haploid and diploid cells carrying plasmid plHl-9, or wild-type and ste4 mutant haploid cells were grown overnight; they were washed and each strain was split into 2 aliquots. One aliquot received a saturating concentration of a factor (1Om6M) and the other did not. After incubation at 24°C for varying times, aliquots of cells were collected by filtration, washed, and resuspended in “inhibitor medium,” containing NaN3 and NaF, to block further membrane traffic. After a 2 hr incubation to allow dissociation of bound a factor, the cells were collected by centrifugation and the remaining cell surface receptors were quantified by a binding assay (see Experimental Procedures for details). As seen in Figure 3, a factor receptors remain at the cell surface in the absence of pheromone but quickly disappear upon addition of a factor. This regulation of a factor receptor internalization is independent of G protein-mediated signal transduction, as diploid and haploid cells show nearly identical results (Figure 3, upper panel), as do isogenie wild-type and ste4 mutant cells (lower panel). Therefore, the regulation of a factor receptor endocytosis and ligand internalization does not depend upon pheromone-

Uncoupling 757

of Ste2p

Down Regulation

and Signaling

% Uptake loo;

;

;

40, 30’ 20Y

“...;:.. x_ -..

70

'-.

'... 60 50 40

".._ "..., . .._ '._. "I... '.., '. '(_.

'...

201

10

'..

'...

'G.. . . . . . . ..__..

30 I

h.

".. .._._..___ diploid + .......___._._. “.pT,.--..... .... .:-: 1.,...,,_____,,,,_, ~::: ,,,,......... @

haploid + , , , .

101 0 1

5

.~__~~ 10

.~_

l~~. 15

, 20

25

30

0 L,~_~~- _ 1

_ ~. 5

time at 24°C Figure netics

2. Nonresponding

Cells

Internalize

_~~ IO

20

'

.

'25

30

time a1524”C u Factor

with Normal

Ki-

Haploid (RH1199, Aste2, circles) and drplord (RH765, squares) yeast transformed with plasmid plHl-9 were grown overnight in YPUAGal medium. Wild-type (RH448. asterisks) and sterile (RH448ste4, triangles) haploid cells were grown overnight in YPUAD medium. Uptake of surface bound %-a factor was measured as described in Experimental Procedures. The results shown above are the average of two experiments each performed in duplicate The average cpm and percentage of total a factor bound at time 0 were 4901 cpm (26% bound), 3721 cpm (21% bound), 3418 cpm (34% bound), 1803 cpm (17% bound), for the haplord, diploid. wild-type, and sre4 mutant cells, respectively.

Binding ‘40 I

‘lo30 -

-..._

20

‘2.. -....._._.,_,7. ;

specific G protein-mediated signal transduction or on any haploid-specific gene product. One difference seen in Figure 3 between wild-type and ste4 mutant cells is that the number of cell surface receptors on wild-type cells began to rise again after about 20 min, and this rise did not occur in the ste4 mutant. The increase is due to new receptor synthesis (Jenness and Spatrick, 1986) emanating from an induction of SE2 by a factor (Nakayama et al., 1985; Hagen and Sprague, 1984). The ste4 mutant does not respond to a factor and therefore there is no rise in cell surface receptors. No differences were seen between the isogenic haploid and diploid strains because the SE2 gene had been placed under galactose (GAL70) control. The Receptor Is Hyperphosphorylated in the Absence of Pheromone-Specific G Protein-Mediated Signal Transduction To test whether the receptor can be hyperphosphorylated in the absence of pheromone-specific G protein-mediated signal transduction, we introduced the plasmid plH2-4 (multicopy) carrying SE2 under galactose control into isogenie haploid and diploid cells, or into an isogenic pair of wild-type and ste4 haploid cells. High production of the receptor was necessary in the following experiments to obtain significant results. The cells were grown overnight at 30% in selective medium with galactose as a carbon source. They were then radioactively labeled with ?S04-’ and 3*P04-3. After a 20 min incubation, the % label was chased with excess Sod-*, methionine and cysteine for 20 additional minutes so most of the labeled receptors would be located at the cell surface. The culture was then divided

_ ( ,_ SW4

10'

__

+

"'~%,_(_

0 1

5

10

Figure 3. Receptor sponding Cells

Down

30

25

time a1524”C Regulation

2o rn Respondrng

and

Nonre-

Haploid (RH1199 transformed with plf-ll-9, Aste2, circles) and diploid (RH765 transformed with plHl-9, squares) cells were grown overnight in YPUAGal medium. Wild-type (RH448, circles) and ste4 mutant (RH448ste4, squares) cells were grown overnight rn YPUAD medium. The cultures were split into two parts and a factor (1O-6 M) was added at time 0 to one aliquot. After incubation for the indicated times, the relative number of cell surface receptors was measured by a binding assay (Experimental Procedures). To average the results from two independent experiments, each performed in duplicate, the binding activity found at 1 mm after a factor addition was set to 100. The broken line is with u factor, the solid line is without (I factor.

into two equal aliquots, and a saturating concentration of a factor (10d6M) was added to one aliquot. After 7.5 min incubation with pheromone (approximately 50% receptor internalization; Singer and Riezman, 1990) the cells were lysed and the extracts were denatured in urea-SDS-containing buffer at 50%. The extractswere then immunoprecipitated with antibodies against the NH2 terminusof Ste2p or with hexokinase antiserum. As a control, cells with a disrupted STEP gene that did not carry a plasmid were labeled and treated in the same manner. The immunoprecipitates were separated on SDS-polyacrylamide gels without reducing agents, as large amountsof reduced IgG heavy chain interfere with the migration of Ste2p in these gels. As can be seen from the autoradiograph in Figure 4, Ste2p was immunoprecipitated from both haploid and

Cell 756

HAP

DIP

-i--+-A

Hex wt

-+ Ste2p

Figure Cells

4. Hyperphosphorylation

ste4

-+A

.!%

of SteZp

Occurs

m NonrespondIng

lsogenic haploid (RH1199, Aste2) or diploid (RH765) cells with or without plasmld plH2-4 and isogenic wild-type (RH270-2B) and ste4 mutant (RH1911) cells carrying plasmid plH2-4 and ste2 mutant (RH1662) cells without plasmid were grown overnight in minimal galactosecontaining medium. The cells were then radiolabeled as described In Experimental Procedureswith30,~‘and3’P04 3.Aftera20minchase wlthcycloheximide.(NH,)LiO,, methionine,andcysteine,afactor(10m6 M) was added to half of the cultures and incubated for an additional 7.5 min. The cells were then lysed, and Ste2p or hexokinase (Hex) was immunoprecipitated and analyzed by SDS-gel electrophoresis and autoradiography. Approximately 2.5 and 1.6 times more 3 cpm were recovered from the haploid than from the diploid in the StePp and hexokinase bands, respectively. HAP, haploid cells with plasmid plH24; DIP, diploid cellswith plasmid plH2-4; wt, wild-typecellswith plasmid plH2-4; ste4, ste4 mutant cells with plasmid plH2-4. -, without a factor; +, with a factor; A, haploid cells wIthout plasmid (upper panel) and ste2 mutant cells (lower panel), each without a factor.

diploid cells, and from wild-type and ste4 haploid cells carrying plasmid, but not from cells with a disrupted STE2 locus. A significantly larger amount of Ste2p was precipitated from the haploid cells than the diploid cells. The lower amount of immunoprecipitable radioactive Ste2p found in the diploid is likely to be due both to a partial repression of STE2 in diploid cells due to the presence of approximately 200 bp of STf2 upstream sequence (from the ATG) in our construct and to a 30%-50% lower labeling efficiency of the diploid cell type as judged from the recovery of immunoprecipitable labeled hexokinase (Figure 4) or carboxypeptidase Y (data not shown). In any event, in response to incubation with a factor there is an apparent increase in the Ste2p signal in all strains, owing to an increase in 32P cpm (see below). In addition, Ste2p migrates with a slightly slower mobility after a factor addition, perhaps owing to the increased phosphorylation state of the receptor. To quantify phosphorylation, the bands were excised from the gels and counted for % and 32P as described in Experimental Procedures. The results from three experiments are shown in Table 1. The basal level of phosphorylation (without a factor) was higher for haploid

cells than for diploid cells. This is not due to the inability to respond to pheromone, because the ste4 mutant cells showed a basal level of receptor phosphorylation that was identical to that found in isogenic wild-type cells. For Ste2p in the isogenic haploid and diploid cells, the average increase in the 32P:35S ratio attributable to a factor is 1.40 times and 1.55 times, respectively. For Ste2p in the isogenie wild-type and ste4 mutant cells this ratio is approximately 1.25 times irrespective of the ste4 mutation. These results suggest that receptor hyperphosphorylation can occur independently of pheromone-specific G proteinmediated signal transduction and other haploid cell-specific functions, The results found above suggest that the processes of hyperphosphorylation in the isogenic wild-type and sfe4 mutant cells are quantitatively similar. To test whether receptor hyperphosphorylation is also qualitatively similar, the isogenic wild-type and ste4 mutant cells were labeled with 32P04m2 under the conditions described above with and without a factor. Ste2p was immunoprecipitated and separated by SDS-polyacrylamide gel electrophoresis, the bands were excised, and the protein was extracted from the gel slices. The radiolabeled Ste2p was then subjected to trypsin digestion and 2-D tryptic phosphopeptide maps were generated. As evident from the autoradiographs of the 2-D maps (Figure 5) the patterns of phosphorylation of Ste2p in wild-type cells and ste4 mutant cells were extremely similar. When a factor was added, the phosphorylation pattern did not change substantially, although the relative intensities of the different spots did change. This is consistent with previous observations (Reneke et al., 1988). The tryptic peptide spots that increased in relative intensity were the same when a factor was added to wild-type or ste4 cells. These results show that hyperphosphorylation of the a factor receptor in response to pheromone is independent of pheromone-specific G protein-mediated signal transduction. Discussion

In this paper, we make three major advances in our knowledge about the relationship between G protein-mediated pheromone signal transduction and the down regulation of the a factor receptor. First, we show that this signal transduction pathway is not necessary for the internalization of the ligand or its receptor. Second, we show that the tripartite G protein that participates in the signal transduction pathway is not necessary for retention of the receptor at the surface in the absence of signal transduction. Third, we show that the hyperphosphorylation of the receptor that occurs concomitant with G protein-mediated pheromone signal transduction is largely independent of this pathway. The first two points were established by measuring the rates of a factor internalization and removal of active receptors from the cell surface. This was performed in two independent situations in which the process of pheromone-specific G protein-mediated signal transduction did not occur. In one situation the signal transduction pathway was blocked by a mutation in the 5 subunit of the G protein. Receptors were expressed from their normal chromo-

Uncoupling 759

Table

of StePp Down Regulation

1. Hyperphosphorylation

Haploid

Diploid

Wild type

ste4

and Signaling

of Ste2p Is Independent

of G Protein-MedIated

Pheromone

Signal Transduction

Experiment

-

+

+/-

Average

1 2 3

0.49 0.42 0.69

0.60 0.75 0.83

1.22 1.70 1.20

1.40

-c 0.33

1 2 3

0.27 0.30 0.38

0.38 0.51 0.59

1 41 1.70 1 55

1.55

f

4 5 6

0.48 0.50 0.57

0 64 0.62 0.68

1 33 1.24 1.19

1.25

-c 0.05

4 5 6

0.50 0.56 0.56

0.68 0.68 0.67

1.36 1.21 1.20

1.26

f

0.15

0.07

Cells transformed with plasmld plH2-4 were labeled with 35S and “P and lysed; Ste2p was lmmunoprecipitated from the extracts and analyzed by SDS-gel electrophoresis as described in the legend to Figure 4. The radioactive bands were localized by autoradiography and excised from the gel; the ass and 3zP dpm were determined In a scintillation counter. Background levels were determlned by extracting an equivalently located and sized gel piece from the Aste2 lane. The 32P levels were at least 112 dpm above background, and the 35S levels were at least 261 dpm above background. The ratios of 32P to % were calculated and are shown for the three experiments without a factor (-) and with a factor (+).

somal locus and their internalization was regulated in an identical fashion to wild-type cells. Since there was still the possibility that the receptor could depend on the presence of other subunits of the pheromone-specific G protein, we investigated pheromone internalization by diploid cells. Diploid cells, which do not express the pheromonespecific G protein (Dietzel and Kurjan, 1987; Miyajima et al., 1987; Whiteway et al., 1989) or any other haploidspecific genes (Herskowitz, 1988, 1989; Sprague, 1990), also showed internalization parameters identical to isogenie haploid cells. These results confirmed that the pheromone-specific G proteins are not necessary, either for a factor internalization or for retention of the receptor at the

cell surface. Therefore, receptor internalization is not a feedback control mechanism that depends on transmission of the pheromone-induced G protein-mediated signal. The a factor receptor has been shown to be hyperphosphorylated on its cytoplasmic tail upon pheromone addition (Reneke et al., 1988; Blumer et al., 1988). Using two different experimental systems, isogenic diploid and haploid cells and isogenic wild-type and ste4 mutant cells, we were also able to show that this hyperphosphorylation does not depend upon G protein-mediated signal transduction. Even though the overall labeling of the receptor (with both 3 and 32P) was somewhat less in diploid cells

Figure 5. 2-D Phosphopeptide Maps of Ste2p from Responding and Nonresponding Cells

wt-

ste4 -

ste4 +

Wild-type (RH270-2B, wt) and ste4 mutant (RH1911, ste4) cells carrying plasmid plH2-4 were grown overnight in minimal galactosecontaining medium and were then labeled with H332P04 as described in Experimental Procedures. Half of the sample received 10e6 M a factor for 7.5 min, then the cells were lysed and Ste2p was immunoprecipitated and purified by SDS-gel electrophoresis. The excised bands were subjected to digestion by trypsin, and the resulting peptides were separated by electrophoresis (horizontal direction) and thin layer chromatography (vertical direction). +, plus a factor; -, no a factor.

Cell 760

than in haploid cells, the increase in the 32Pto ?S ratio due to addition of a factor was indistinguishable. Measuring this ratio in the experiment effectively corrected for any variations due to the different incorporation of radioactive precursors or different recoveries during cell lysis and immunoprecipitation. In the wild-type cells and in the ste4 cells, Ste2p showed identical behavior for both basal levels of phosphorylation and for hyperphosphorylation caused by pheromone addition. 2-D tryptic maps of the phosphopeptides derived from Ste2p before and after hyperphosphorylation in wild-type or ste4 cells looked very similar, indicating that the phosphorylated sites are similar in the mutant and wild-type cells. In fact, no consistent differences in phosphorylation sites could be found when comparing the results of several experiments. Our results demonstrate that hyperphosphorylation of the receptor is not caused by a direct feedback regulation that could be invoked owing to the activation of kinases known to be involved in the pheromone transduction pathway (Fields, 1990; Marsh et al., 1991, for review). The kinases responsible for receptor hyperphosphorylation do not need to be activated by G protein-specific signaling nor are they haploid-specific genes. This does not rule out the possibility that some protein kinases involved in the pheromone signaling pathway, for example Ste7p and Stel 1p, which are expressed in diploid cells (Rhodes et al., 1990; see Marsh et al., 1991) could be involved in receptor hyperphosphorylation, but they would not need to be activated by pheromone. What controls receptor internalization and hyperphosphorylation? Recently, another receptor-dependent signaling pathway that does not seem to depend on the G proteins has been postulated (Jackson et al., 1991). This pathway is responsible for mating partner discrimination. It could be that this signaling pathway has a feedback loop that controls receptor hyperphosphorylation and internalization. Another possibility, that we prefer, is that the receptor undergoes a conformational change upon pheromone binding. This change would allow the receptor to interact productively with the pheromone-specific G protein, expose receptor sites to a protein kinase or mask them from a protein phosphatase, and allow the receptor to interact with the endocytic machinery. This putative conformational change could also be important for the G protein-independent signaling pathway. There are several possibilities for this putative conformational change. There could be some rearrangement of the transmembrane domains of the receptor upon binding of a factor. This could in turn lead to a change in conformation of the parts of the protein exposed to the cytoplasm. Another possibility would be that the receptors dimerize or aggregate upon a factor addition. In ouropinion, thiswould also require a conformational change of the receptor, because a factor is too small to crosslink receptors physically. To date, only indirect evidence exists for receptor aggregation (Jenness and Spatrick, 1986; Blumer et al., 1988). A receptor system with some common features with the pheromone system of S. cerevisiae is the f3-adrenergic receptor system (see Benovic, 1988 for review). In both

cases, receptors that span the membrane seven times are coupled to G proteins and seem to be phosphorylated or hyperphosphorylated and removed from the plasma membrane by endocytosis upon ligand binding. The f3-adrenergic receptor G protein is coupled to an adenylate cyclase, and it seems that most of the effects of the ligand, except homologous desensitization, are transduced through this pathway (Benovic et al., 1988). Homologous desensitization seems to depend on phosphorylation (Bouvier et al., 1988), which still takes place in mutant S49 lymphoma cell lines incapable of CAMP-mediated signaling (Strasser et al., 1986). These results are very similar to those we presented, except that the phosphorylation that occurs in their mutant S49 lymphoma cellsonly seems to be a subset of what is normally found on the receptor. Phosphorylation was found only on serine residues instead of on both serine and threonine residues. It would be interesting to know if endocytosis of the 8-adrenergic receptor occurs normally in these mutant cell lines. Regulated endocytosis of another receptor, the insulin receptor, may be controlled by autophosphorylation or tyrosine kinase activity and thus require some signal transduction (Hari and Roth, 1987; McClain et al., 1987; Russell et al., 1987). One major drawback of these studies, however, is that insulin receptor mutantswere analyzed. When receptor tyrosine kinase activity was abolished by mutagenesis, receptor internalization no longer occurred. An alternative explanation for this essentially negative result is that this mutation modified the receptor into a conformation that could not be endocytosed. Similar studies of the epidermal growth factor receptor led to conflicting results and conclusions (Chen et al., 1987,1989; Honegger et al., 1987; Felder et al., 1990). In the present study, we have presented a positive result showing that the regulation of a factor receptor internalization does not depend on the G protein-mediated pheromone signal transduction pathway. These experiments did not introduce modifications into the receptor itself and therefore are not subject to the same criticism as the above experiments. From our data we cannot speculate on whether the hyperphosphorylation of the receptor is a necessary requirement for receptor internalization. Experiments to address this question await the identification of the hyperphosphorylated sites and their modification, or the identification and inactivation of the kinases involved. Experimental

Procedures

Plasmids, Strains, Reagents, and Media Plasmid plHl-9 was constructed by replacing the 2.6 kb Hindlll fragment of pGAL-HO (CEN4-ARS1. URA3, GAL-HO; Herskowitz and Jensen, 1990) with the 1.65 kb Htndlll fragment of pAB510 (Burkholder and Hartwell, 1965) in the proper orientation to place STEP under control of the GAL70 promotor. plH2-4 was constructed by inserting sequentially the 365 bp GAL70 promotor and the 1.65 kb STE2 sequences between the EcoRl and Hindlll sites of pSEY8 (2 tr, URA3; Emr et al., 1966). Standard molecular biology techniques were used (Maniatis et al., 1982). The following yeast strains were used: RH448 (MATa, ura3, leu2, his4, lys2, gal, bad-l), RH446ste4 (MATa, ura3, leu2, ste4::LEUP, his4, lys2, gal, bad-l) (created by one-step gene replacement [Rothstein, 19631 using Pstl cut ~4-121; Whiteway et al., 1969) RH151-7B (MATa, ura3, leu2, his4), RH151-7Blys2 (spontaneouslys2derivativeofRH151-7B).RH1199(RH151-7Blys2withaste2::

Uncoupkng 761

of Ste2p

Down Regulation

and Signalmg

LEU2 gene replacement using pUSTE203; Nakayama et al., 1988 and a barl::LYSP gene replacement using plasmid pEK-3; Dulic and Riezman, 1990) RH765 (diploid cell derived by HO-induced switching of RH151-79; Herskowitzand Jensen, 1990) RH270-29 (MATa, ura3, leu2, his4, lys2, bad-l), RH1911 (RH270-29 with a ste4::LEUP gene replacement using ~4-121) RH1662 (RH270-29 with a steP::LEUP gene replacement using plasmid pUSTE203). Cellswere grown in oneof two rich media, YPUAD or YPlJAGal(l% yeast extract, 2% peptone, 40 Kg/ml uracil, 40 Kg/ml adenine sulfate, 3% galactose) or in minimal media lacking uracil (Dulic et al., 1990) with either 2% glucose (SD-ura) as a carbon source or 3% galactose (SGal-ura) as a carbon source. SGallnoPS-ura was SGal-ura with KCI substituted for potassium phosphate and NH&I substituted for (NH&SO4 and was buffered with 7 mM succinate (pH 5.8). SGall IowPS-ura was SGallnoPS-ura plus 50 uM each of KH,PO, and (NH&SO,. Anti-peptide antibodies recognizing the NH2 terminus of Ste2p were raised against the synthetic peptide, SDAAPSLSNLFYDPTK, after its coupling to Keyhole limpet hemocyanin (KLH) as described (Ternynck and Avrameas, 1976; Louvard et al., 1983). Peptide (2.4 mg) was coupled to 4 mg of KLH. Conjugated KLH (300 ug) was used for the initial intradermal injection and 250 ug was used for subsequent boosts. %-a factor was produced and purrfred as described (Dulrc et al., 1990) from culture supernatants of biosynthetically labeled RH449 (MATa, ura3, leu2, his4, lys2, barl-7) cells transformed with plasmid pDA6300 to overproduce biologically active a factor. Protein A-Sepharose was from Pharmacia, Emulsifier-Safe, Soluene-350, and HIONIC-FLUOR were from Packard. N”-tosyl-L-argrnine methylester hydrochloride (TAME) and benzamidine were purchased from Merck, pepstatin A from Sigma Chemical Co., sodium metavanadate and sodium pyrophosphate from Fluka, and sodrum orthovanadate from Aldrich. H2%0, (carrier free) and Hz3*P0, (carrier free) were obtained from Amersham. Yeast extract and peptone were obtained from Gibco.

D Factor Internalization and Receptor Clearance Assays a factor internalization was measured as described (Dulic et al., 1990; Zanolari and Riezman, 1991). Strains RH1199 and RH765 (both transformed with plasmid plHl-9) were inoculated from saturated precultures grown in SD-ura medium into YPUAGal medium and grown overnight at 24% to approximately l-2 x lO’cells/ml. The cells were washed, resuspended in ice-cold YPUAGal medium, and incubated wrth %S-a factor for 40 min on ice. The cells were collected by centrifugation and resuspended in 24% YPUAGal and incubated for various trmes at 24OC. Aliquots were removed into pH 1.I or pH 6 buffer and washed on filters to differentiate between internalized and bound 35S-a factor, respectively. The filters were dried at 80% and counted in a Packard 1900TR scintillation counter using Emulsifier-Safe scintillation fluid. 35S-a factor internalization by strains RH448 and RH448ste4 was measured as described above, except that YPUAD was substituted for YPUAGal. Receptor down regulation was measured as described (Jenness and Spatrick, 1986) with minor modifications. Erther strains RH765 and RH1199 carrying plasmid plHl-9, or strains RH448 and RH448ste4 without plasmid were grown overnight as described above. Each strain was then treated rn the following manner. Cells (2 x log) were harvested by centrifugation, split into two alrquots, and resuspended in 200 ml YPUAGal medium per aliquot containing 10 mM TAME. The cells were preincubated for 15-30 min at 24OC rn a shaking water bath, then a factor (1Om6 M final concentration) was added to one 200 ml aliquot. At different time points, 20 ml aliquots were collected by filtration on nrtrocellulose filters (HA, 0.45 mm, Millipore), washed with 2 x 2 ml inhibitor medium (YPUAGal containing 10 mM each NaF, NaN3, and TAME), and placed in 30 ml of inhibitor medium at 24%. After 130-150 min, the cells were harvested by centrifugation and resuspended in 0.5 ml of inhibitor medium. The relative number of cell surface receptors was measured by a binding assay performed in duplicate. Aliquots (0.1 ml) of cells were incubated with %-a factor for 30 min at 24OC, then collected by filtratron. washed with inhibitor medium on Whatman GFlC filters, dried, and counted as above. Backgrounds (subtracted) were determined by performing the binding assay in the presence of 2 x 10m5 M unlabeled a factor.

Measurement of Receptor Hyperphosphorylation The assay for receptor hyperphosphorylation was performed as modified from Reneke et al. (1986). Strains RH1199, RH765, RH270-28, and RH1911 carryrng plasmid plH2-4 were grown overnight in SGalura. Cultures of strains RHI 199 and RH1662 without plasmid were prepared identically, but were supplemented with 40 uglml uracil. Cells (1 x 10’) were collected by centrifugation, washed with distilled H,O, and resuspended in 20 ml of SGalllowPS-ura medrum. After 5 hr shaking at 30°C, the cells were harvested by centrifugation, washed with SGallnoPS-ura medium, and resuspended in 2 ml of the same medium. H2%01 (400 FCi) and Hs3’POa (1 mCi) were added, and the cells were incubated with shaking for 20 min at 30%. Cycloheximide (25 uglml frnal concentration), (NH&SOn(2 mM), methionine, and cysteine (each at a concentration of 20 uglml) were added, and the cells were incubated an additional 20 min. The samples were then divided Into two 1 ml aliquots and one of these received a factor to a final concentration of 10 6 M. After an additional 7.5 min incubation, the cells were added to 0.1 ml of a 10x stop solution (4 rnM each sodium meta and orthovanadate, 0.1 M NaN3, 0.1 M NaF, 4 mM EDTA, 10 mM benzamidine, 5 uM pepstatin A, 100 mM sodium pyrophosphate) and quickly frozen in liquid NY. After thawing, the cells were collected by centrifugation, resuspended in 1 ml breaking buffer (9 M urea, 40 mM Tris-HCI [pH 6.81, 0.1 mM EDTA, 140 mM 2-mercaptoethanol), and broken by vortexing 4 x 1 min in the presence of glass beads. After removing the samples from the beads, SDS was added to a concentration of l%, and the extracts were heated to 50°C for 10 min to solubilrze Ste2p. The extracts were diluted lo-fold with immunoprecipitation buffer (50 mM Tris-HCI [pH 7.51, 150 mM NaCI, 5 mM EDTA, 1% Tnton X-l 00,l mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate) and insoluble material was removed by centrifugation (Sorvall SS-34 rotor) at 20,000 rpm for 30 min. Ste2p, hexokrnase. or carboxypepbdase Y were then immunoprecipitated as described (Gasser et al., 1982) except that protein A-Sepharose beads (Pharmacia) were substituted for S. aureus cells, and the immunoprecipitate was eluted from the beads using breaking buffer without 2.mercaptoethanol by heating to 50°C for 10 min. The immunoprecipitates were analyzed by SDS-gel electrophoresis (Laemmlr, 1970). The relevant proteins were localized by exposure of the dried gels to Kodax X-OMAT AR film at -70°C using a screen. The radioactive bands were excised from the gels, and the gel slices were solubilized in 2 ml of Soluene 350 for 3.5 hr at 50%. The samples were decolorized with isopropanol and HZ02 at 40°C, according to the instructions from Packard and 10 ml of HIONIC-FLUOR was added. Double counting for 32P and ?S with corrections for quench was achieved in the Packard 1900 TR using the program installed by the manufacturer for this purpose. For 2-D tryptic mapping of the phosphorylated receptor, RH270-2B or RH1911 cells transformed with plasmid plH2-4 were grown overnight in SGAL-ura medium at 30°C. Cells (5 x 10’) were washed with distilled water, resuspended in 10 ml of SGAUlowP-ura medium, and incubated for 4.5-5 hr. The cells were then washed, resuspended in SGAUnoP-ura, and Incubated with 1 mCi of H,32P0, for 40 min at 30°C. After a 7.5 min exposure to 10 6 M a factor, the phosphorylated StePp was immunoprecipitated as described above and purified by SDS-gel electrophoresis. 2-D tryptic phosphopeptide maps were obtained as described (Boyle et al., 1991). The receptor was localized by exposure of the dried gel to film and excised by tuning with scissors. After removal of as much of the paper as possible, the gel slice was allowed to swell in 0.7 ml of 0.05 M NH,HCO, for 1 min. The gel slice was then cut into small cubes (1 mm) and 0.3 ml of NH,HCOJ, 50 ug bovine serum albumin, and 10 ul of 10% SDS were added. After overnight incubation with rotation at 3O”C, the mix was spun at top speed in an Eppendorff centrifuge, and the supernatant was removed. 8-mercaptoethanol was added to 5% final concentration, and the sample was heated to 50°C for 10 min. Trichloroacetic acid was added to 17.2% final concentration, and the precipitate was recovered after 1 hr incubation on ice followed by a 10 min spin at 15,000 rpm (SS34 rotor). The pellets were washed with 1 ml -20°C ethanol, with 1 ml -20°C ethanol:ether (1 :l, vollvol) and air dried. The dried pellet was resuspended in 100 ul performic acid and incubated on ice for 1 hr. H20 (0.8 ml) was added, and the sample was frozen. After drying, the resulting pellet was resuspended in 150 ul of 0.05 NH,HCO, (pH 8) and digested with 25 ug TPCK-treated trypsin (Sigma Chemical Co.) for 5 hr at 37%. An additronal20 ug trypsin was added and incubatron

Cell 762

contmued for another 4 hr. After extensive washes wrth HZ0 to remove the bicarbonate, the digest was drssolved in pH 4.72 electrophoresis buffer (Boyle et al., 1991) and spotted onto 20 x 20 cm DC-cellulose plates (Merck). Electrophoresis was performed for 1 hr at 1000 V, and the plate was then air dried overnight. Ascending chromatography was then performed for about 6.5 hr in phospho chromatography buffer (Boyle et al., 1991). The dried plates were then exposed to preflashed X-ray film for 1 to 5 days. Acknowledgments We would like to thank Scott Emr (University of California at San Diego), Rob Jensen (Johns Hopkins Medical School), Lee Hartwell (University of Washington at Seattle), Naoki Nakayama(Tokyo University), and Debra Barnesand JeremyThorner(UniversityofCaliforniaat Berkeley). We would also like to thank Vivian MacKay (Zymogenetics, Seattle) for plasmids, lsabelle Howald for construction of plasmids plH1-9 and plH2-4, Fabienne Crausaz for excellent technical assistance, and Jack Rohrer, Linda Hicke, and Alan Munn for critically reading the manuscript. A special thanks goes to Stefano Ferrari for his advice and help in generation of 2-D phosphopeptide maps. Thus work was supported by a grant from the Swiss National Science Foundation to H. R. and by the Canton of Basel-Stadt. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

February

4, 1992; revised

September

22, 1992

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