Diagram of the potential phosphorylation sites in sst2 receptor splice variants. The structure of the carboxyl-terminal portion of the rat sst2A and sst2B receptors are shown schematically. The serine and threonine residues mutated to alanine in this study are indicated in yellow and red, respectively. We previously showed by phosphopeptide mapping that phosphorylation occurs on residues in the third intracellular and C-terminal regions of the sst2A receptor (Hipkin et al., 2000; Elberg et al., 2002). 

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Context 1

... and Gurevich, 2006; DeWire et al., 2007; Moore et al., 2007). Peptide mapping has shown that SS14 stimulates the phosphorylation of the sst2A receptor on the CT and IC3 loop in pituitary (Hipkin et al., 2000), pancreatic acinar (Elberg et al., 2002), and CHO cells (Q. Liu and A. Schonbrunn, unpub- lished observations). Furthermore, Ser residues were phosphorylated to a much greater extent than Thr residues in GH pituitary cells (Hipkin et al., 1997). Consistent with our previous studies, we found that mutation of all the Ser/Thr residues in the CT and IC3 loop of sst2A prevents both basal and SS14-stimulated receptor phosphorylation in CHO cells. Two new lines of evidence further indicate that receptor phosphorylation occurs mostly on Ser residues. First, phosphoamino acid analysis of the wild-type receptor isolated from SS14-treated cells showed 4 times as much 32 P incor- poration into phosphoserine as into phosphothreonine. Second, phosphorylation was reduced to a much greater extent in the Ser Ϫ mutant than in the Thr Ϫ mutant. However, because partial mutation of receptor phosphorylation sites in the Ser Ϫ and Thr Ϫ mutants did not prevent agonist stimu- lated phosphorylation on remaining residues, the regulation of these mutant receptors can be used to reveal the functional importance of the intact phosphorylation sites. Desensitization of GPCRs, defined as a reduction in receptor signaling after pre-exposure to agonist, is a complex process that can involve either reduced coupling efficiency between cell surface receptors and the intracellular signaling machinery or a loss of plasma membrane receptors due to receptor endocytosis. Internalization and desensitization of the sst2A receptor both occur within a few minutes of SS14 treatment (Hipkin et al., 1997; Elberg et al., 2002). The simultaneous occurrence of these events makes it impossible to study receptor uncoupling in isolation. Nonetheless, we minimized the contribution of receptor trafficking to our desensitization assays by using a 5-min SS14 pretreatment and by measuring the subsequent receptor response in a cell-free assay in which receptor trafficking could not occur. In addition, because desensitization is often manifest as a change in EC 50 instead of or in addition to a change in maximal inhibition, we carried out full dose-response curves for SS14 when testing for receptor uncoupling. Finally, we monitored the rate of receptor internalization rather than just the extent of internalization after a new steady-state was achieved. Together, these experimental paradigms, which differ from most previous studies of sst2A receptor regulation, were critical for quantitating receptor desensitization and endocytosis and for monitoring internalization and uncoupling sepa- rately in our internalization-deficient mutants. The mutant receptors used in this study were all well expressed at the cell surface, and they inhibited adenylyl cyclase in response to nanomolar concentrations of SS14, demonstrating that the introduced mutations did not produce general changes in receptor function. Therefore, the fact that internalization was markedly slowed and receptor uncoupling was blocked in the phosphorylation negative Ser Ϫ / Thr Ϫ mutant indicates that sst2A receptor phosphorylation is required for both efficient endocytosis and uncoupling. However, different phosphorylated regions of the receptor were necessary for these regulatory events. Mutation of only Thr or only Ser residues did not prevent desensitization, although receptor phosphorylation was reduced in both cases. Thus, each group of residues can support receptor uncoupling, indicating that there is substantial structural flexibility in the desensitization mechanisms. In contrast, mutation of the Thr, but not the Ser residues, inhibited receptor internalization similarly to that seen with the full mutant, showing that the Thr residues alone are critical for efficient agonist induced receptor endocytosis despite the fact that Thr residues are phosphorylated to a much lesser extent than Ser residues. Two comparisons demonstrate that receptor endocytosis and uncoupling are regulated independently. First, WT and Thr Ϫ receptors are desensitized similarly, even though the former is internalized 5 times more rapidly. Second, although the Thr Ϫ and the Ser Ϫ / Thr Ϫ mutant receptors internalize at a similar slow rate, the Thr Ϫ mutant is desensitized normally, whereas the Ser Ϫ / Thr Ϫ mutant does not desensitize. Interestingly, our conclusion that endocytosis and uncoupling of the sst2A receptor are independent events contrasts with the results obtained with the sst2B receptor, for which desensitization was found to be a consequence of receptor endocytosis (Beaumont et al., 1998). The sst2A and sst2B splice variants differ in the sequence and length of their C termini: the sst2B receptor ends after residue 332; therefore, it is missing five Ser and five Thr present in the C terminus of sst2A (Fig. 1). In fact, a recent report shows that although the sst2A receptor was rapidly phosphorylated upon agonist stimulation of colonic adenocarcinoma cells, phosphorylation of the sst2B receptor was not detectable (Holliday et al., 2007). The observed differences in the regulation of the two sst2 splice variants is likely to have important biological consequences in tissues, such as the brain and the gastrointestinal tract, in which alternative splicing of sst2 receptor mRNA occurs (Cole and Schindler, 2000). Both uncoupling and internalization have been proposed to depend on the binding of arrestins to phosphorylated, activated GPCRs (Gurevich and Gurevich, 2006; DeWire et al., 2007; Moore et al., 2007). In fact, GPCRs have been classified on the basis of the affinity and duration of their association with arrestins after agonist activation and during the internalization process (Oakley et al., 2000). We previously showed that ␤ -arrestin2 becomes tightly associated with the wild-type sst2A receptor after agonist stimulation (Liu et al., 2005). Here, we demonstrate that the same is true for ␤ -ar- restin1, in agreement with other studies (Brasselet et al., 2002; Tulipano et al., 2004). Thus, agonist treatment triggers the recruitment of both arrestins to the plasma membrane receptor, and the stable association of the receptor-arrestin complex is maintained during endocytosis and intracellular trafficking. The continuous association of the sst2A receptor with both arrestins during internalization shows that this receptor belongs to the class B GPCRs. Our new results with phosphorylation-deficient mutant receptors demonstrate that only the Thr residues in the C-terminal domain of the sst2A receptor are required for the formation of a stable arrestin-receptor complex: Ser residues in the C terminus and IC3 loop are dispensable, despite the fact that these Ser are heavily phosphorylated. Thus, arrestin binding to the sst2A receptor requires particular phosphorylated residues in the C terminus: these residues are not redundant or in- terchangeable. The close correlation between the ability of different receptor mutants to internalize rapidly and their association with arrestins further indicates that arrestins have an important role in sst2A receptor endocytosis. However, internalization was not completely blocked in the Thr Ϫ and Ser Ϫ / Thr Ϫ receptors, which do not recruit arrestins; internalization continued at 10 to 20% the rate of the wild-type receptor. Thus, receptor phosphorylation is not an absolute requirement for sst2A internalization, but rather it greatly accelerates the process. These results suggest two mechanisms of sst2A receptor internalization: a rapid process, with a half-time of about 5 min that is dependent on receptor phosphorylation and stable arrestin association, and a slower process, with a half-time of 40 min, that still occurs with the nonphosphory- lated Ser Ϫ / Thr Ϫ receptor mutant in the absence of arrestin recruitment. We do not know whether the trafficking pathways and mechanisms used for the internalization of the WT, Thr Ϫ , or Ser Ϫ / Thr Ϫ mutant receptors are the same or different. However, the ability of sucrose to block the endocytosis of both the Thr Ϫ and Ser Ϫ / Thr Ϫ mutant receptors as effectively as the wild-type receptor indicates that internalization always occurs by a clathrin-mediated pathway. Whereas our results indicate that receptor-arrestin associ- ation is required for efficient sst2A endocytosis, Brasselet et al. (2002) previously concluded that arrestins were not involved in the internalization of the sst2A receptor. The stron- gest support provided for this conclusion was an immunofluorescence experiment showing that the dominant-negative ␤ -arrestin-1(319-418) did not block receptor internalization. However, our results suggest that the dominant-negative ␤ -arrestin may only inhibit receptor endocytosis rather than prevent it completely. In our study, endocytosis of the slowly internalizing Thr Ϫ and Ser / Thr Ϫ mutants are both readily detected in similar immunofluorescence experiments, pre- sumably because fluorescence microscopy is particularly sensitive to the intense signal produced by aggregated receptors in intracellular vesicles. Thus, the results from our studies and those of Brasselet et al. (2002) are not inconsistent once two rates of receptor endocytosis with different arrestin requirements are recognized. Overall, the data support the conclusion that rapid endocytosis of the sst2A receptor after agonist stimulation occurs by an arrestin-dependent mechanism, although the receptor can internalize at a slow rate in an arrestin-independent manner. Unexpectedly, we found that the Ser Ϫ receptor internalized even more quickly than the WT receptor. Because the Ser Ϫ mutant was phosphorylated on Thr in response to SS14 stimulation, perhaps phosphorylation of the critical Thr residues in the C-terminal domain of sst2A is facilitated in ...

Context 2

... We previously showed that agonist treatment stimulates the rapid phosphorylation of the sst2A receptor on two domains: the IC3 and the CT (Hipkin et al., 2000; Elberg et al., 2002). To investigate the functional importance of receptor phosphorylation, we generated three mutant receptors with different Ser/Thr-to-Ala substitutions in these two receptor domains (Fig. 1): 1) Ser Ϫ in which all serine residues in the IC3 loop and the CT region were mutated to Ala, 2) Thr Ϫ in which all threonine residues in the CT region were mutated to Ala, and 3) Ser Ϫ / Thr Ϫ in which Ala was substituted for all serine and threonine residues in the IC3 loop and CT region. All receptor constructs contained a triple HA epitope tag at the amino terminus of the receptor to permit ready quantitation of plasma membrane receptors by ELISA (Liu et al., 2005). Addition of this extracellular epitope tag does not affect any of the functional properties of the receptor, including ligand binding affinity, receptor internalization, or inhibition of cyclic AMP production (Liu et al., 2005). All three sst2A receptor mutants were expressed at the cell surface to similar levels, responded to nanomolar concentrations of agonist, and coupled normally to inhibition of adenylyl cyclase (see below), demonstrating that the introduced mutations did not lead to general perturbations in receptor function. Because both receptor internalization and signaling are known to be affected by the level of cell surface receptor expression (Cole et al., 2001; Dunigan et al., 2002), we screened multiple clonal cell lines stably expressing either wild-type or individual mutant receptors to identify clones that contained a similar density of cell surface receptors. Plasma membrane sst2A receptors were detected by ELISA with an antibody that recognized the amino-terminal HA epitope (Liu et al., 2005). Because our ELISA protocol was carried out with nonpermeabilized cells, the HA epitope was accessible to antibody in the medium only when the receptor was correctly expressed on the cell surface (Liu et al., 2005). In contrast, the R2-88 antibody, directed to the carboxyl- terminal region of sst2A (Gu and Schonbrunn, 1997), was unable to detect plasma membrane sst2A receptors in this ELISA unless the cells were first permeabilized with detergent (data not shown). To quantitate receptor density, a standard curve of surface receptors as a function of cell protein was constructed for individual CHO-K1 cell clones expressing either the wild- type sst2A receptor (Fig. 2, top) or different mutant receptors (data not shown). Using a linear regression fit, we calculated the relative receptor density for each cell line, and then we selected cell clones that expressed similar levels of cell surface receptor (Fig. 2, bottom). In multiple independent experiments with the selected cell clones, the density of the Ser Ϫ , Thr Ϫ , and Ser Ϫ / Thr Ϫ mutant receptors were 126 Ϯ 38% ( n ϭ 3), 152 Ϯ 25% ( n ϭ 5), and 83 Ϯ 19% ( n ϭ 4) that of the cell clone expressing wild-type receptors. These results demonstrate that the wild-type and mutant sst2A receptors are expressed to similar levels in the selected CHO-K1 cell lines. The experiments described below show that all the receptors are also well coupled to adenylyl cyclase inhibition (Fig. 11; Table 1), confirming that the mutant receptors are present on the cell surface in a functional form. ceptor Mutants. Our previous peptide mapping experiments showed that the sst2A receptor was phosphorylated in the C- terminal and third intracellular loop domains after SS14 stimulation (Hipkin et al., 2000; Elberg et al., 2002). To determine whether receptor phosphorylation was reduced in the mutant sst2A receptors as expected and to estimate the relative importance of Ser and Thr phosphorylation, we metabolically labeled cells with 32 PO and then we incubated them without or with 100 nM SS14 for 15 min. Receptors were then purified by lectin affinity chromatography followed by immunoprecipitation with an antibody to the amino-terminal HA epitope. The amount of radiolabel incorporated into purified receptors was quantitated using a PhosphorImager, and then it was corrected for receptor concentration as determined in the same samples by immunoblotting. Figure 3A shows the results of a representative experiment, and Fig. 3B summarizes the data from three independent experiments. Basal phosphorylation was reduced in both the Ser Ϫ and Thr Ϫ mutants, and it was completely eliminated in the Ser Ϫ / Thr Ϫ mutant. After SS14 stimulation, the amount of 32 P incorporated into the Ser Ϫ receptor was reduced by 65 Ϯ 10% (mean Ϯ S.E.M.; n ϭ 3) compared with the wild-type receptor. However, radiolabeling was decreased by only 14.4 Ϯ 3.0% in the Thr Ϫ mutant. Mutating both Ser and Thr residues abolished SS14-stimulated receptor phosphorylation. These results indicate that SS14 stimulates sst2A receptor phosphorylation on both Ser and Thr. However, the Ser residues constitute the major sites for sst2A receptor phosphorylation after agonist treatment. The amino acids phosphorylated in the WT and mutant receptors were next characterized directly by phosphoamino acid analysis of purified receptors prepared from SS14- treated cells (Fig. 3C). Quantitation of the radiolabel incorporated into phosphoserine and phosphothreonine residues showed that in the wild-type receptor 80% of the 32 P was associated with phosphoserine and 20% was associated with phosphothreonine. In contrast, and as expected, 32 P was exclusively present in phosphoserine in the Thr Ϫ mutant, whereas phosphothreonine was labeled in the Ser Ϫ mutant. Although the acid hydrolysis method used in these experiments does not quantitatively convert all of the radioactivity present in a phosphoprotein to phosphoamino acids (Kamps, 1991), the results again show that SS14 stimulates sst2A receptor phosphorylation primarily on Ser residues. In summary, the metabolic labeling experiments with 32 PO 4 demonstrate that mutation of all the Ser and Thr residues in the C terminus and third intracellular loop of the sst2A receptor abrogates both basal and SS14-stimulated sst2A receptor phosphorylation, consistent with our previous peptide mapping data identifying these as the only phosphorylated domains in sst2A (Hipkin et al., 2000; Elberg et al., 2002). Serine residues constitute the primary sites for agonist-stimulated receptor phosphorylation, and threonine residues are phosphorylated to a much lesser extent. Deficient Mutant Receptors. Figure 4 (top) shows the rate of internalization of wild-type and mutant sst2A receptors after stimulation with a saturating concentration (100 nM) of SS14. The calculated half-times determined in multiple independent experiments are summarized in Table 1. The Ser Ϫ receptor consistently demonstrated rapid internalization that was about 2 times faster than the wild-type receptor. In contrast, although the Thr Ϫ and Ser Ϫ / Thr Ϫ mutants still internalized after agonist stimulation, their rates of internalization were markedly reduced: the Thr Ϫ mutant receptor internalized about 5 times more slowly and the Ser Ϫ / Thr Ϫ mutant internalized about 8 times more slowly than wild-type sst2A (Table 1). Figure 4 (bottom) shows the dose dependence for SS14- induced receptor internalization. A summary of the values for the EC 50 and maximal internalization in three independent experiments is shown in Table 1. The Ser Ϫ mutant showed the same sensitivity to SS14 as the wild-type receptor, although the extent of Ser Ϫ receptor internalization was slightly higher. The Thr Ϫ and Ser Ϫ / Thr Ϫ mutants were 3- to 4-fold less sensitive to SS14-induced internalization, and the extent of receptor internalization at 30 min was less than that for the wild-type receptor. The observation that SS14 stimulated receptor internalization with similar potencies in the WT and mutant receptors shows that the introduced mutations had minimal effects on agonist binding per se. Furthermore, the rapid internalization of the Ser Ϫ receptor demonstrates that phosphorylation of serine residues in the IC3 and CT domains is not essential. In contrast, the impaired rate of internalization of the Thr Ϫ and the Ser Ϫ / Thr Ϫ mutant receptors shows that phosphorylation of the threonine residues in the carboxyl-terminal domain is required for rapid sst2A receptor ...