Internalization and Homologous Desensitization of the GLP-1 Receptor Depend on Phosphorylation of the Receptor Carboxyl Tail at the Same Three Sites

Christian Widmann1, Wanda Dolci and Bernard Thorens

Institute of Pharmacology and Toxicology, University of Lausanne CH-1005 Lausanne, Switzerland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molec-ular mechanisms controlling desensitization and internalization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Agonist binding to heterotrimeric G protein-coupled receptors triggers several events that are thought to result from ligand-induced changes in receptor conformation. First, dissociation of G{alpha}-subunits from their ß{gamma}-partners leads to activation of the intracellular signaling pathways. Usually, after this first event, there is a rapid desensitization of the receptors. This is observed by a decrease in the maximum, and/or an increase in the EC50, of the dose-response curve for production of intracellular second messengers in a second exposure of the cells to the agonist. Also occurring with a rapid kinetics is the internalization of the receptor-ligand complex by endocytosis through coated pits.

The molecular mechanisms underlying homologous and heterologous desensitization of these receptors usually involve phosphorylation of specific residues by different kinases. For instance, for the ß2-adrenergic receptor, heterologous desensitization can be induced by activation of protein kinase A (PKA) or protein kinase C (PKC), an effect that requires the presence of a PKA/PKC consensus site in the third intracellular loop of the receptor (1, 2, 3, 4). On the other hand, homologous desensitization results from receptor phosphorylation by G protein-coupled receptor-specific kinases (GRKs) (5), which form a family of structurally related isoforms (6). This phosphorylation takes place in the serine/threonine-rich C tail of the receptor (4) and induces association of the receptor with ß-arrestins (7, 8, 9, 10). This interaction prevents further activation of G proteins after agonist binding to the receptor and is thought to form the basis for the observed desensitization process.

Internalization of G protein-coupled receptors rapidly follows agonist binding and has initially been proposed to be involved in receptor desensitization (11, 12). More recently, studies have shown, on one hand, that desensitization could be suppressed by removing the GRK phosphorylation sites of the ß2-adrenergic receptor without impairing receptor internalization (4, 13). On the other hand, mutants unable to be internalized were still desensitized (14, 15). This indicated that internalization of the receptor-ligand complex and homologous desensitization were independent processes. This situation needs to be reappraised, however, in light of work demonstrating that internalization of the m2 muscarinic acetylcholine receptor can be facilitated by overexpression of GRK2 and that expression of a dominant negative mutant of this kinase decreases the rate of receptor internalization (16). Also, endocytosis of an internalization-resistant mutant of the ß2-adrenergic receptor could be induced by overexpression of GRK2, which phosphorylated the receptor (17). These two studies therefore suggest that phosphorylation by GRKs, if not totally required, may nevertheless participate in the endocytosis process. A role for ß-arrrestin in facilitating the internalization of GRK-phosphorylated ß2-adrenergic receptor has further been demonstrated (18), and ß-arrestin appears to function as a clathrin adaptor in receptor endocytosis (19). Together, the above evidence indicates that, although for some receptors desensitization and internalization are two events that can proceed independently of each other, phosphorylation of receptors by GRKs and consequent binding of ß-arrestins may participate in both phenomena.

Internalization of single transmembrane receptors such as those for transferrin, low-density lipoproteins, insulin, and epidermal growth factor require a tyrosine residue present in a tight-turn-forming motif of the sequence NPXY (20, 21). Internalization of other membrane proteins, such as the T lymphocyte CD3 antigen (22), the IgGFc receptor (23), or the glucose transporter GLUT4 (24), depends in great part on the presence of a dileucine internalization motif. For G-coupled receptors, however, no specific internalization motif has been described, although mutations of single amino acids may prevent internalization in some instances. This is the case for tyrosine 326 of the ß2-AR receptor (14), for three threonine residues of the cytoplasmic tail of the m3 muscarinic acetylcholine receptor (25), and for specific lysine residues of the yeast {alpha}-pheromone receptor (26).

The glucagon-like peptide-1 receptor is a G-coupled receptor expressed by pancreatic ß-cells (27). Binding of GLP-1 activates the adenylyl cyclase pathway, which ultimately results in the strong potentiation of glucose-induced insulin secretion (28, 29). We previously described that homologous and heterologous (PKC-induced) desensitization of the receptor strictly correlated with receptor phosphorylation in the last 33-amino acid segment of the receptor C tail (30). Furthermore, we showed that the PKC phosphorylation sites were four serine doublets present in this segment of the receptor and that serine doublet at position 431/432 was the major phosphorylation site (31). In this study we characterize the sites phosphorylated after agonist binding and demonstrate that these phosphorylation sites are required not only for homologous desensitization but also for internalization of the receptor-ligand complex.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of GLP-1 to its receptor expressed in insulinomas and fibroblasts (30) or in COS cells (see below) induces phosphorylation of the receptor by a PKA- and PKC-independent mechanism. Previous experiments demonstrated that, in fibroblasts, phosphorylation took place in the carboxyl-terminal 33-amino acid segment of the receptor and that this phosphorylation strictly correlated with desensitization of the cAMP response (30).

Here, to identify the phosphorylated amino acids, we first performed a phosphoamino acid analysis of the phosphate-labeled receptor expressed in fibroblasts or COS cells. Figure 1Go shows that phosphorylation of the GLP-1 receptor expressed in both cell types occurs only on serine residues. To determine which serines were phosphorylated in the carboxyl-terminal segment of the receptor, we constructed several deletion and point mutants of the receptor. Figure 2Go shows the sequence of the receptor cytoplasmic tail and of the different mutants tested. Ten serines are present in the region of the receptor that contains the phosphorylation sites and that extends from serine 431 to the carboxyl end at position 463. Eight of these serines are present as four doublets, and two individual serines are at positions 461 and 463. In a first set of experiments, carboxyl-tail deletion mutants were transiently expressed in COS cells. Binding affinity and coupling to production of cAMP were identical for the truncated mutants and the wild type receptor (not shown). After radiolabeling with radioactive orthophosphate, the cells were exposed to GLP-1 for 15 min and lysed, and the receptor was immunoprecipitated and analyzed by gel electrophoresis. Figure 3AGo shows phosphorylation of the wild type receptor and of deletion mutants {Delta}CT451 and {Delta}CT444. No phosphorylation of the receptor, however, could be detected in the {Delta}CT441 and {Delta}CT431 mutants. This suggests that phosphorylation takes place at least on the last three serine doublets but not on the doublet at position 431/2. Identical results were obtained with the same truncation mutants stably transfected in fibroblasts (Ref. 30 and not shown), therefore indicating no cell type differences in receptor phosphorylation.



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Figure 1. Phosphoamino Acid Analysis of the GLP-1 Receptor

Phosphoamino acid analysis was performed on immunoprecipitated and gel-purified receptors prepared from fibroblasts (leftpanel) or COS cells (right panel) prelabeled for 2 h with [32P]orthophosphate and exposed for 15 min to 10 nM GLP-1. The left part of each panel shows the receptor immunoprecipitated from cells exposed (+) or not (-) to GLP-1 and purified by SDS-gel electrophoresis. The phosphoaminoacid analysis is on the right part of each panel. The position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) is indicated.

 


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Figure 2. Sequence of the GLP-1 Receptor C-Tail Mutants

The top line shows the sequence of the wild type receptor carboxy-terminal cytoplasmic tail (position 412 to 463). Sequence of the truncated mutants and of the point mutants is indicated below the wild type sequence; only the mutated amino acids are shown.

 


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Figure 3. Phosphorylation of the Receptor Mutants

A, The wild type or truncated mutants of the GLP-1 receptor were transiently transfected in COS cells. After labeling with [32P]orthophosphate, the cells were exposed (+) or not (-) for 15 min to 10 nM GLP-1, and the receptors were immunoprecipitated and separated on SDS-polyacrylamide gels. Phosphorylation is observed in the wild type and the {Delta}CT451 and {Delta}CT444 receptor mutant but not in the other two truncation mutants. B, The wild type receptor or point mutants having a single serine doublet left intact and the others mutated to alanines were transiently transfected in COS cells, and phosphorylation after agonist stimulation was assessed as in panel A). The three distal serine doublets were phosphorylated but not that present at position 431/432.

 
To identify the phosphorylation sites, we next evaluated the phosphorylation of four mutants in which a single serine doublet was left intact while the other three were mutated to alanines. These mutants were then transiently transfected in COS cells, and their phosphorylation after exposure to GLP-1 was assessed as described above. Figure 3BGo shows that the mutants 451SS, 444SS, and 441SS were phosphorylated after GLP-1 binding while mutant 431SS was not. These data thus indicate that the last three serine doublets are phosphorylated after agonist binding to its receptor. This is in agreement with the results obtained with the deletion mutants. Furthermore, because no phosphorylation can be detected in the 431SS mutant, we conclude that serines at positions 461 and 463, which are retained in this mutant, are not phosphorylated.

To determine whether phosphorylation was correlated with receptor homologous desensitization, we established fibroblast cell lines expressing the wild type or the different mutant receptors under the control of the metallothionein promoter, as described (31). Figure 4Go (left panel) shows that desensitization of the wild type receptor could be measured by a decrease in the Vmax for cAMP production by about 50%. In these cells only a small shift in the EC50 for cAMP production was observed (from 2 to 5.8 nM). Mutation of all three serine doublets to alanine generated a receptor in which desensitization was almost completely suppressed (Vmax = ~90% of nondesensitized receptor) (Fig. 4Go, right panel). We similarly tested cell lines expressing mutant receptors with only a single doublet or two doublets mutated to alanine and evaluated the Vmax for cAMP production after desensitization. Figures 5Go and 7Go show a summary of the data obtained for all the clones tested. For the wild type receptor, Vmax was reduced to 58% of the nondesensitized receptor. With a single doublet mutated to alanine, Vmax was reduced to 75–80% of nondesensitized receptor. Double and triple mutants showed the same reduction of Vmax after desensitization to ~90% of the nondesensitized receptor. These data indicate a correlation between the extent of phosphorylation and the decrease in Vmax after desensitization. Phosphorylation on at least two serine doublets is required to observe intermediate desensitization, and maximal desensitization is only obtained when phosphorylation occurs at all three identified sites.



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Figure 4. Desensitization of the GLP-1 Receptor and of the Phosphorylation Negative Mutant

The wild type receptor or the 431SS mutant in which the three distal serine doublets were mutated to alanines were stably transfected in Chinese hamster lung fibroblasts. The dose-dependent production of cAMP in response to increases in GLP-1 concentrations was measured in untreated cells (control) or cells preexposed for 15 min to 10 nM GLP-1. Wild type receptor-expressing cells (left panel) show a marked reduction in the maximal production of cAMP after agonist-induced desensitization whereas this desensitization was considerably reduced in cells expressing the phosphorylation mutant (right panel).

 


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Figure 5. Decreased Desensitization of Phosphorylation Site Mutants

The maximal production of cAMP (Vmax) of cells expressing the different receptor point mutants was measured after preexposure of the cells to GLP-1 as described in Fig. 4Go. Vmax for the wild type receptor is decreased by ~43% after desensitization; it is decreased by ~20–25% in mutants containing only two phosphorylation sites and by ~10% in mutants with only a single or no phosphorylation sites left unmutated. The results are expressed as the mean ± SEM for n = 3–7.

 


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Figure 7. Functional Modifications in Receptor Phosphorylation Site Mutants

The figure shows the sequence of the cytoplasmic tail of the receptor. The sites phosphorylated in response to GLP-1 binding (homologous phosphorylation, this study) or in response to PKC activation [heterologous desensitization (31 )] are indicated by arrows. The mutated sequences are presented below the wild type sequence, and the corresponding modifications of desensitization and internalization are indicated on the right. Desensitization is expressed as the percent of maximal cAMP production by desensitized compared with nondesensitized receptors. Mutations of single serine doublets lead to intermediate levels of desensitization whereas mutations of two or three serine doublets almost completely suppress desensitization. Internalization is expressed as the percent of bound [125I]GLP-1, which becomes internalized after 5 min of initiating endocytosis. Mutations of single serine doublets lead to reduction of internalization, but the effect of the 451AA mutation is less marked than that of the other mutations. Removal of two serine doublets leads to a more extensive reduction of internalization. Complete suppression of internalization, however, requires mutations of the three doublets. The differential effect of double and triple mutations on receptor desensitization and internalization suggests that both mechanisms, although dependent on receptor phosphorylation, may be regulated by different molecular mechanisms.

 
We previously demonstrated that agonist binding induces a fast (t1/2 = 2–3 min) internalization of the ligand-receptor complex (32). We further showed that truncation of the cytoplasmic tail at position 444 generated a receptor that was no longer internalized (30). This receptor could, however, still be desensitized after activation of PKC which phosphorylates the receptor mostly on serines 431/432. These preliminary experiments thus indicated that the sequence containing the homologous desensitization phosphorylation sites was required for agonist-induced internalization. As no known internalization motifs are present in this region, we evaluated the role of the identified phosphorylation sites in receptor internalization by measuring the extent of endocytosis of [125I]GLP-1 bound by the different receptor mutants at 0 and 5 min after the cells were warmed. Preliminary measurement of internalization kinetics with the wild type (32) and various mutant receptors indicated that internalization proceeded linearly over this period of time. Figure 6AGo shows the acid wash-resistant [125I]GLP-1 measured at 0 and 5 min. A nondissociable component representing about 25% of initial binding is present at time 0 in all clones except in that expressing the 444/451AA mutant for which this value, for an unknown reason, is 40%. The inset in Fig. 6AGo shows the differences between the acid wash-resistant [125I]GLP-1 at time 0 and 5 min, which represents the peptide internalized over this period of time. Internalization is maximal for the wild type receptor; it is reduced when any single phosphorylation site is mutated to alanines; it is further decreased when two sites are mutated and is suppressed in the absence of phosphorylation sites (see Fig. 7Go). It appears, however, that mutation of position 451/452 has a smaller effect in reducing internalization than mutation of the two other sites. Also, mutation of the three sites has a distinctly more pronounced effect on blocking internalization as compared with the double mutant. These two features indicate different correlation between phosphorylation and internalization and phosphorylation and desensitization. Figure 6BGo shows, by western blot analysis, the redistribution of the receptors to light density vesicles (endosome-enriched) after exposure of the cells to GLP-1. Whereas the wild type receptor content of light density vesicles is increased and that of the heavy density vesicles (plasma membrane-containing) is decreased upon exposure of the cells to GLP-1, no such redistribution can be seen with the 431 SS mutant. This shows, in an independent manner, that the suppression of the three phosphorylation sites indeed prevents agonist-induced receptor internalization.



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Figure 6. Internalization of Wild Type and Mutant Receptors

A, [125I]GLP-1 was bound to fibroblasts expressing the wild type or the mutant receptors for 4 h at 4 C. The cells were then placed at 37 C for 0 or 5 min. Acid wash-resistant radioactivity was measured and expressed as percent of total initial specific binding. For all receptor forms, the results presented are mean ± SEM of three experiments each performed in triplicate. The inset shows the differences in acid wash-resistant [125I]GLP-1 at 0 and 5 min and represents the amount of peptide internalized over this period of time by each mutant. Suppression of one phosphorylation site reduces the rate of internalization. This rate is further reduced in double mutants and suppressed in the triple mutant. Mutation of doublet 451/452 (451AA) appears to have a lower effect in reducing internalization as compared with the other phosphorylation sites. B, Internalization of the wild type or 431SS receptors was measured after exposure of the receptor-expressing cells to 10 nM GLP-1 for 15 min. Endocytosis was assessed as the redistribution of the mature form of the receptor from a plasma membrane-enriched (H) to an endosome-enriched (L) vesicle fraction obtained by sucrose density fractionation of cell homogenates prepared before (0) or after (15 ) addition of the peptide to the cells for 15 min. Upper and lower arrows point to the mature and core-glycosylated forms of the receptor, respectively. In wild type receptor-expressing cells, GLP-1 binding induces an increase in receptor in the endosomal compartment and a decrease from the plasma membrane-enriched fraction. No such effect could be observed with the phosphorylation-negative receptor (431SS). The core-glycosylated, intracellular form of the receptor is not affected by GLP-1 binding.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we identified the sites phosphorylated after GLP-1 binding to its receptor as being three serine doublets present in the last segment of the receptor C tail. We showed that phosphorylation of the receptor was responsible for most of the observed homologous desensitization. The same three identified phosphorylation sites were also required to control receptor endocytosis. However, studies with phosphorylation site mutants suggest that desensitization and internalization may be controlled by different molecular mechanisms. These data are summarized in Fig. 7Go.

Binding of GLP-1 to its receptor leads to activation of adenylyl cyclase and the production of cAMP. This signaling pathway can be attenuated by homologous desensitization but also after activation of PKC (heterologous desensitization). We previously showed that both forms of desensitization were additive in increasing the EC50 and decreasing the Vmax for cAMP production (30). Phosphorylation of the receptor induced by homologous and heterologous desensitization was also additive, suggesting that different amino acids were phosphorylated. PKC-induced desensitization of the receptor involved phosphorylation on the four serine doublets present in the last 32 amino acids of the receptor C tail (31). Here we provide evidence that the last three serine doublets of the C tail are the sites phosphorylated upon agonist binding. These sites thus overlap those phosphorylated by PKC activation. As these sites are doublets of serines, the additivity of the phosphorylation obtained in homologous and heterologous desensitization may be explained by phosphorylation on different serine residues at each of the doublets located at positions 441/442, 444/445, and 451/452 and by phosphorylation of doublet 431/432 uniquely by PKC. This is further supported by the fact that PKC induces only a marginal internalization of the receptor (30) compared with that induced by agonist binding, which requires receptor phosphorylation on the distal three sites. We have previously shown that neither PKC nor PKA were responsible for phosphorylation of the receptor after agonist binding (30). Receptor-specific kinases of the GRK family may thus be involved in this process. Studies in progress will indicate which, if any, of the so far identified GRKs participate in this phosphorylation process.

Implication of receptor phosphorylation in homologous desensitization was studied after transfection of the wild type receptor or of mutants thereof in fibroblasts using an expression vector containing the metallothionein promoter. This promoter permits expression of the receptor at a low level (2000–4000 receptors per cell), comparable to the expression of the endogenous receptor in different insulinoma cell lines (27). This is essential to perform desensitization experiments. Indeed, in the presence of high surface expression, as obtained in COS cells, the basal production of cAMP after a first stimulation of the cells with GLP-1 is too high and does not permit generation of a significant dose-response curve in a second exposure to the peptide. With the presently used fibroblast cell lines, we could study the effect of mutating the three phosphorylation sites together or in different combinations. Our results indicated that desensitization could be mostly, but not completely, suppressed by mutating the three sites. This indicates that phosphorylation plays a major role in inducing homologous desensitization but also suggests that additional modifications may also participate in the observed desensitization. These results are comparable to those obtained with the {Delta}CT431 receptor (30), which is not phosphorylated but still partially desensitized. Interestingly, however, we observed that the extent of desensitization was dependent on the number of phosphorylation sites present. When one site was removed, desensitization was intermediate, and when two sites were mutated desensitization was as impaired as with the triple mutant. Homologous desensitization is thought to be mediated by ß-arrestin binding to phosphorylated receptors. Although the exact mechanism for GLP-1 receptor desensitization is not known, our data suggest that phosphorylation at multiple, adjacent sites is required to generate high affinity binding sites for ß-arrestins.

Internalization of receptor-ligand complexes is an essential aspect of the function of G protein-coupled receptors. It is required for dissociation of the ligand from its receptor but also for resensitization of the receptor (15, 33), probably by dephosphorylation of the receptor by phosphatases encountered in the transit through the endosomal compartment. Signals for internalization of this class of receptors are not well defined. In the present study, we showed, using point mutants, that removing the three phosphorylation sites led to a complete suppression of receptor internalization. Mutation of one or two sites, however, led to rates of internalization that were intermediate between that of the wild type receptor and that of the triple mutant. The different phosphorylation sites, however, appear to contribute differentially to receptor endocytosis. This is especially noticeable for the site at position 451/452, which has a significantly smaller effect in reducing the internalization rate than mutation of the other sites (see Fig. 7Go). Altogether, these data show that there is thus a strict correlation between internalization and phosphorylation. However, this correlation is qualitatively different from that observed between phosphorylation and desensitization. This is evident especially for the double and triple mutants. Whereas these two classes of mutants are equally resistant to desensitization, there is a marked difference in the ability of the double mutants to be internalized, at a reduced but still significant rate, compared with the triple mutant, which is not internalized at all. Also, mutation of site 451/452 has a much lower effect in reducing internalization compared with mutation of either of the two other phosphorylation sites. No such differential effect on desensitization can be observed with the single mutants. Together, these data indicate that phosphorylation of the receptor at the identified sites by a kinase activated after agonist binding is responsible for two events: homologous desensitization and receptor internalization. The exact contribution of each phosphorylation site to both mechanisms appears to be, at least in part, distinct, suggesting that the molecular basis for the control of receptor desensitization and endocytosis is different.

In pancreatic ß-cells, glucose-induced insulin secretion can be potentiated by activation of receptors linked to the adenylyl cyclase or the phospholipase C pathways. Activation of muscarinic receptors by carbachol strongly stimulates the insulin-secretory response but, at the same time, it induces a strong desensitization of the GLP-1 receptor (C. Widmann and B. Thorens, unpublished observations). By its poor ability to induce receptor internalization, PKC may desensitize the receptor for a longer period of time compared with homologous desensitization if internalization is primarily required for receptor resensitization. This may be of functional significance in the integration by ß-cells of different signals modulating insulin secretion, in particular in the postprandial state when cholinergic and gluco-incretin (GLP-1, glucose-dependent insulinotropic polypeptide) signals converge to the ß-cells to stimulate their secretory activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
COS cells and Chinese Hamster Lung (CHL) fibroblasts were cultured as described (34). Clone 5 is a CHL fibroblast cell line stably transfected with the rat GLP-1 receptor cDNA (32, 34). Transformation of COS cells and generation of stable CHL transformants were performed as described earlier (34). The number of cell surface-expressed receptors in stably transfected fibroblasts expressing the wild type or mutant forms of the receptor has been established by saturation binding experiments (2000–4000 receptors per cell) and was published previously (31).

Mutagenesis
The different GLP-1 receptor mutants used in this study are described in Fig. 2Go. The deletion mutant {Delta}CT431 mutant was described previously (30). The other mutations were generated by PCR amplification, as described (31, 35), and each mutant was verified by DNA sequencing. The mutant GLP-1 receptor cDNAs were subcloned in the pcDNA-3 vector (Invitrogen, Leek, The Netherlands) and in the pmlMTIi vector (30). The cDNAs subcloned in the pcDNA-3 and pmlMTIi vectors are under the control of the cytomegalovirus and metallothionein promoters, respectively.

Desensitization of the GLP-1-Induced cAMP Production
Desensitization was assessed as described earlier (30), by comparing the dose-response curves of cAMP production as a function of increasing GLP-1 concentrations for control cells and cells pretreated for 15 min with 10 nM GLP-1 at 37 C.

Phosphorylation and Phosphoamino Acid Analysis
GLP-1-induced phosphorylation of the wild type or mutant GLP-1 receptors expressed transiently in COS cells was assessed as described earlier (30). Briefly, COS cells were transiently transfected with the different receptor cDNA constructs by the diethylaminoethyl-dextran technique. For radiolabeling, cells were incubated for 2–3 h in the presence of 500 µCi/ml of [32P]orthophosphate. GLP-1 (10 nM) was then added to the cells for 15 min, and the cells were quickly washed in ice-cold PBS (8 g/liter NaCl, 0.2 g/liter KCl, 1.44 g/liter Na2HPO4·2 H2O, 0.2 g/liter KH2PO4, pH 7.4). The cells were detached from the culture dishes after a 10-min incubation with 1.5–2 ml of PBS, 1 mM EDTA and collected in an Eppendorf tube. The cells were lysed in PBS containing 1% Triton X-100, 5 mM EDTA, 1 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 25 mM NaF, and 1 mM NaVO4 for 10 min at 4 C. Immunoprecipitation of the receptor and analysis by gel electrophoresis were then performed exactly as described (31).

Phosphoamino acid analysis of the gel-purified receptor was performed by TLC using a HTLE 7000 electrophoresis system (C.B.S. Scientific Company, Del Mar, CA) according to the manufacturer’s protocol and Copper et al. (36).

Internalization of Receptor-Ligand Complex
Measurements of ligand-receptor internalization were performed as previously described (32). Briefly, for evaluation of [125I]GLP-1 endocytosis, the radiolabeled peptide (400 pM) was first bound at 4 C for 4–6 h, after which the cells were washed with ice-cold HBSS containing 20 mM HEPES, pH 7.4, and returned to 37 C for the indicated periods of time. The cells were then washed with the HBSS buffer and lysed with 0.2 N NaOH/1% SDS, and the radioactivity was counted (total binding). Alternatively, a duplicate set of cells was washed and incubated for 2 min in an acidic solution (50 mM glycine, 150 mM NaCl, pH 3) to remove surface-bound radioactivity. The cell-associated radioactivity that remained was considered to represent the internalized peptide. A fraction of surface-bound [125I]GLP-1 could not be removed at pH 3 before incubation at 37 C (~30%).

Analysis of GLP-1 receptor redistribution to endosomal compartments after GLP-1 binding was studied exactly as described (32). Briefly, cells expressing the wild type receptor or different mutants were exposed for 15 min to GLP-1 at 37 C. The cells were then treated with 500 µg/ml Concanavalin A and lysed in a hypotonic lysis buffer (1 mM Tris-HCl, pH 7.4, 2 mM EDTA). The cells were then scraped with a rubber policeman, and the total cell lysate was loaded on a discontinuous sucrose gradient consisting of 4 ml 60% sucrose, 4 ml 38% sucrose, and 4 ml 15% sucrose all made up in 20 mM Tris-HCl, pH 7.4. After centrifugation at 112,000 x g for 1 h at 2 C in a Beckman SW40 Ti rotor, the membrane fractions at the 25–38% sucrose interface (endosomal fraction) and 38–60% sucrose interface (plasma membrane fraction) were collected and analyzed for the presence of the receptor by Western blot analysis using receptor-specific antibodies (32).


    FOOTNOTES
 
Address requests for reprints to: Bernard Thorens, Insitute of Pharmacology and Toxicology, University of Lausanne, 27, rue du Bugnon, 1005 Lausanne, Switzerland.

B.T. was supported by a Career Development Award from the Swiss National Science Foundation. This work was supported by Grant 31–30313-90 from the Swiss National Science Foundation.

1 Present address: National Jewish Center for Immunology and Respiratory Medicine, Pediatrics Department, 1400 Jackson Street, Denver, Colorado 80206. Back

Received for publication January 14, 1997. Revision received March 11, 1997. Accepted for publication April 16, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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