©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Glycosylation and Intracellular Trafficking for the Long and Short Isoforms of the D Dopamine Receptor (*)

(Received for publication, July 13, 1995; and in revised form, September 25, 1995)

C. Simone Fishburn (1) Zvulun Elazar (2) Sara Fuchs (1)(§)

From the  (1)Departments of Chemical Immunology and (2)Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The D(2) dopamine receptor exists in two alternatively spliced isoforms, ``long'' and ``short'' (D and D), which differ by 29 amino acids in the third cytoplasmic domain. The functional differences between these two isoforms are still obscure. We have performed pulse-chase studies on the D and D receptors expressed in CHO cells in order to follow the post-translational processing of the two isoforms. Both isoforms are present in three post-translational states: a newly synthesized protein, a partially glycosylated product, and a fully glycosylated mature 70-kDa receptor. However, the processing to the mature receptor differs between the two isoforms. First, the D receptor is processed to the mature 70-kDa species faster than the D receptor. Second, at 20 °C the D isoform is fully processed to the 70-kDa species, whereas the D isoform persists in its partially processed 45-kDa state. Finally, a significant portion of the D receptor remains in its partially processed form in an intracellular compartment and does not reach the plasma membrane. These results give rise to the suggestion that the difference observed between the two alternatively spliced isoforms of the D(2) receptor may lie in their post-translational processing and intracellular trafficking.


INTRODUCTION

The existence of multiple dopamine receptors was for many years postulated to underlie the diverse behavioral and biochemical properties associated with dopaminergic neurotransmission and dopamine receptor activation(1) . These receptors were known to belong to the G protein-linked receptor superfamily and were divided according to their ability to stimulate (D1) or inhibit (D2) adenylate cyclase activity (2) . Cloning studies of the past few years have revealed the complexity of dopamine receptors through the discovery that there are at least two genes encoding D1-type receptors (D and D) (3, 4, 5, 6) and three genes encoding D2-type receptors (D(2), D(3), and D(4))(7, 8, 9) . This heterogeneity was found to be yet further diverse by the identification of two alternatively spliced isoforms of the D(2)(10, 11, 12) and subsequently D(3)(13) receptor subtypes. In both cases, alternative splicing yields functional long and short receptors, which differ in the putative third cytoplasmic loop by the presence or absence of 29 (D(2)) or 21 (D(3)) amino acids. In this receptor superfamily, the third cytoplasmic domain has been highlighted for its involvement in G protein-coupling(14, 15) .

Although functional studies on the long and short D(2) receptors (D and D) in various cells have revealed their ability to couple to a range of second messenger pathways(16, 17, 18) , a clear functional difference between the short and long alternatively spliced isoforms has yet to be identified. Recent studies have raised the possibility that the D and D receptors may be differently phosphorylated (19, 20) . In addition, it has been demonstrated that the two isoforms can display differential G protein interaction(21) . Here, we have explored the possibility that the long and short D(2) receptor isoforms may differ in their post-translational modifications. The fact that D(2)-type dopamine receptors are glycosylated was established by early photoaffinity labeling studies that utilized glycosidase treatment to demonstrate the presence of oligosaccharides on D(2)-like receptors(22, 23, 24) . In addition, a previous study in our lab examining the biosynthesis of the rat D receptor showed it to be synthesized as a protein of approximately 35-kDa and to undergo processing to yield first a 45-kDa and then a 70-kDa species(25) .

In this paper, we have studied the manner in which the two receptor isoforms, expressed in CHO (^1)cells, are progressively glycosylated as they travel through the cell's post-translational trafficking pathway. We have examined for the first time the post-translational processing of the short isoform of the D(2) receptor and present evidence that the D and D receptors are differently glycosylated and are subject to different patterns of intracellular trafficking.


EXPERIMENTAL PROCEDURES

Materials

[S]Methionine (>3000 Ci/mmol) was purchased from Amersham Corp. Proteinase K and bovine serum albumin were purchased from Sigma. Protein A-agarose (Immobilized rProtein A) was purchased from Repligen (Cambridge, MA). Endo H and N-glycanase (PNGase F) were purchased from New England Biolabs, Inc. (Beverly, MA). All cell culture media were from Life Technologies, Inc.

Methods

Antibody Preparation

The D(2)-specific peptide SPPERTRYSPIPPS (amino acids 288-301) derived from the predicted sequence of the murine D(2) dopamine receptor (26) was synthesized in the laboratory of peptide synthesis of the Weizmann Institute of Science by the solid phase method of Merrifield. Specific anti-peptide antibodies were elicited and purified on a bovine serum albumin column as described previously(27) .

CHO Cell Culture and Transfection

Two cDNA clones, encoding the murine D and D receptors, respectively, were subcloned into the eukaryotic expression vector pcDNAI/Neo (Stratagene). The expression constructs contained identical coding and noncoding sequences for the D and D receptor isoforms, except for the alternatively spliced 87 base pairs in the putative third cytoplasmic loop. CHO cells were transfected with 20 µg of D or D receptor DNA using electroporation (250 V, 500 µF). Stable transfectants were selected in the presence of 500 µg/ml G418 (Life Technologies, Inc.) and were subcloned by limiting dilution. Positive receptor-expressing cell lines were identified by Northern analysis and [^3H]spiperone binding. Comparative analysis of D and D receptor properties was performed on cell lines expressing similar levels of receptor (approximately 400 fmol/mg of protein). Cells were cultured in Ham's F-12 medium containing 10% fetal calf serum, 30 µg/ml gentamicin sulfate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 250 µg/ml fungizone and were maintained in a humidified incubator (5% CO(2)) at 37 °C.

[S]Methionine Labeling

CHO cells stably transfected with the D or D receptor (5 times 10^6/10-mm plate) were washed twice with phosphate-buffered saline and then incubated for 1 h in methionine-free Dulbecco's modified Eagle medium with 5% dialyzed fetal calf serum. Cells were labeled for 15 min except where indicated otherwise, in the presence of 150 µCi of [S]methionine in 2 ml methionine-free medium. For pulse-chase experiments, cells were washed twice and then incubated in complete medium at 37 °C and 95% humidity for the time indicated. Incubations at other temperatures were performed in water baths with control plates being incubated in parallel in a 37 °C water bath. Following the labeling or the chase, cells were placed on ice. Solubilization of membranes, immunoprecipitation of D(2) receptors, and gel electrophoresis were performed as described previously(25) .

Endoglycosidase Treatment

Following immunoprecipitation, samples were denatured and eluted from the beads by incubation in 30 µl of denaturing buffer (0.5% SDS, 1% beta-mercaptoethanol) for 30 min at 37 °C. Treatment with 10 units/µl Endo H was performed for 1 h at 37 °C in the presence of 50 mM sodium citrate (pH 5.5). Treatment with 10 units/µl N-glycanase (PNGase F) was performed for 1 h at 37 °C in the presence of 50 mM sodium phosphate (pH 7.5) and 1% Nonidet P-40. Reactions were terminated by the addition of sample buffer, and samples were electrophoresed on polyacrylamide gels as above.


RESULTS

Dand DReceptors Are Each Present in Three Different Post-translational Forms

In order to investigate whether the long and short isoforms of the D(2) dopamine receptor may differ in their post-translational processing, we have performed pulse-chase studies in CHO cell lines stably expressing comparable levels of the murine D and D receptors. Cells were given a 15-min pulse with [S]methionine, and the respective D or D receptors were immunoprecipitated using a specific anti-D(2) receptor antibody. In accordance with previous results obtained for the rat D isoform(25) , two specific bands were observed at approximately 45 and 39 kDa. The D receptor was found to be present in two corresponding forms at 43 and 37 kDa (Fig. 1a). This difference in apparent molecular mass between the long and short D(2) receptors can be explained by the 29 amino acids in the putative third cytoplasmic loop that differentiate between the two receptor isoforms. When the cells were given a 3-h chase with full medium following the [S]methionine pulse, both D and D receptors were observed as high molecular mass bands of approximately 70 kDa, bearing the somewhat hazy appearance characteristic of heavily glycosylated proteins (Fig. 1b). These three forms, at 39/37, 45/43, and 70 kDa, were shown specifically to represent the D(2) receptor because they were not present in untransfected CHO cells (Fig. 1) or when immunoprecipitation was performed in the presence of the relevant peptide (see Fig. 4and 5).


Figure 1: Immunoprecipitation of [S]methionine-labeled D and D dopamine receptors from stably transfected CHO cell lines. Cells were incubated with [S]methionine for 15 min, and immunoprecipitation was carried out immediately (a) or following a 3-h chase in full medium (b) as described under ``Materials and Methods.'' Samples were run on 7.5-15% gradient SDS-polyacrylamide gels treated with Amplify (Amersham Corp.) for intensification of the [S]methionine signal and autoradiographed. The arrows indicate the specific 39- and 45-kDa D, 37- and 43-kDa D, and the 70-kDa D and D products.




Figure 4: N-glycanase treatment of D receptors in transfected CHO cells. Cells were given a 15-min pulse with [S]methionine and then chased for 3 h in full medium. Following immunoprecipitation with the D(2)-specific antibody in the presence or absence of inhibiting D(2)-peptide or with D(3) peptide for control, samples were split into two and incubated in the presence or the absence of N-glycanase. The arrows indicate specific 39-, 45-, and 70-kDa D products.



Interestingly, although a portion of the 45-kDa form of the D receptor persisted after 3 h, the corresponding 43-kDa D receptor species could no longer be observed. Densitometric scanning performed on results from several experiments showed that more than 20% of the labeled D receptor remained at 45 kDa after 3 h. This phenomenon was shown to be a feature of the D(2) receptor itself, rather than a cell-specific property arising through the transfection process, because it was seen in several different CHO cell lines expressing the murine D(2) receptors. In addition, the difference between the long and short D(2) receptor isoforms was observed by metabolic and photoaffinity labeling studies on the rat D and D receptors expressed in CHO cells, in which the D isoform produced signals at 45 and 70 kDa, whereas the D isoform produced a signal only at 70 kDa(25) . Thus, in both the mouse and rat D(2) receptors, the D receptor appears to undergo full processing to the mature 70-kDa species, whereas a significant portion of the D receptor remains in the partially processed 45-kDa form.

We have previously shown that the newly translated full-length D and D receptors run at 39 and 37 kDa, respectively, on SDS-polyacrylamide gel electrophoresis (28) and have demonstrated that the 45-kDa D receptor species represents a partially processed form of the receptor(25) . To determine whether this rise in apparent molecular mass is the result of N-linked glycosylation, metabolic labeling was performed as for Fig. 1a, and the immunoprecipitated products were treated with Endo H. As can be seen from Fig. 2, this treatment results in the complete loss of the 45- and 43-kDa D and D species, respectively, and the concomitant enrichment of the 39 and 37-kDa bands, showing that the higher molecular mass band represents a partially processed product arising from N-linked glycosylation of the newly translated receptor.


Figure 2: Endo H treatment of D and D receptors in transfected CHO cells. Metabolic labeling was performed as for Fig. 1a, and following immunoprecipitation, samples were denatured and split into two to be incubated in the presence or the absence of Endo H. Products were electrophoresed as described. The arrows indicate the specific 39- and 45-kDa D or 37- and 43-kDa D products.



Because the 45-kDa and 43-kDa bands consistently appeared stronger than their 39-kDa and 37-kDa precursors, respectively, it seemed that the majority of the newly translated receptor proteins may undergo N-linked glycosylation during the 15-min [S]methionine labeling period. In order to investigate whether there may be differences between the long and short D(2) receptors in the generation of this glycosylation intermediate and to see whether the newly translated proteins could be observed in the absence of the partially glycosylated products, cells were given only a 5-min incubation with [S]methionine before immunoprecipitation, and the results compared with those following a 15-min incubation. The results in Fig. 3show that even after a pulse of only 5 min, both the 45- and 43-kDa D and D bands were present, and it was not possible to observe the newly translated products in the absence of the partially glycosylated proteins. In addition, after only 5 min of labeling, the partially glycosylated bands were four times stronger than their 39- and 37-kDa precursors, as determined by densitometric scanning. The ratio of partially glycosylated to newly translated receptor was found to be the same for both D and D receptors and not to differ between the 5- and 15-min labeling times, suggesting that the initial N-linked glycosylation occurs efficiently and rapidly for both the long and short receptor isoforms.


Figure 3: Time course of labeling of D and D receptors in transfected CHO cells. Cells were incubated with [S]methionine for 5 or 15 min, and immunoprecipitation was then carried out as described. The arrows indicate the specific 39- and 45-kDa D or 37- and 43-kDa D products.



To investigate whether the 70-kDa product arises as a result of glycosylation of the smaller molecular mass forms of the receptor, pulse-chase labeling was performed as for Fig. 1b, and the immunoprecipitated products were treated with N-glycanase (PNGase F). This treatment resulted in the disappearance of the 70-kDa band, and the appearance of a 39-kDa band (Fig. 4). It should be noted that the 45-kDa D product, still present after 3 h of chase, is also reduced to 39-kDa by this treatment and that all three bands represent specific D(2) receptor forms, because they are not observed when immunoprecipitation is performed in the presence of inhibiting peptide. The presence of an irrelevant peptide based on the D(3) receptor sequence does not remove the 70- and 45-kDa bands. Similar results were obtained for the D receptor (data not shown). Because N-glycanase reduces the 70- and 45-kDa products to the molecular mass of the newly translated receptor, it seems that following rapid N-linked glycosylation of the 39-kDa D translation product to the 45-kDa glycosylation intermediate, a subsequent slower N-linked glycosylation occurs, producing the fully glycosylated mature 70-kDa receptor. Moreover, it can be inferred that the carbohydrate modifications on the mature protein occur exclusively through N-linked and not at all through O-linked glycosylation. Thus it appears that the 39-, 45-, and 70-kDa D receptor species and their 37-, 43-, and 70-kDa D counterparts represent forms of the protein present at progressive stages in the receptors' post-translational glycosylation pathway.

Dand DReceptors Are Differently Processed

To examine whether the rate of processing to the 70-kDa mature species may differ between the long and short D(2) isoforms, we have performed pulse-chase studies on D- and D-transfected cell lines expressing equivalent levels of receptor (Fig. 5). Using 15- and 60-min chase periods, it could be seen that for both receptor isoforms, the newly translated product (39-kDa D and 37-kDa D) persists for 15 min following labeling but has been almost completely converted to the subsequent glycosylated forms of the receptor by 60 min. Interestingly, as early as 15 min following the pulse, a significant amount of the newly synthesized D receptor has been converted to the mature 70-kDa form, and within 1 h it has all been fully processed. By contrast, the D receptor shows a very small amount, if any, of the mature 70-kDa form after 15 min of chase and appears to undergo a slower conversion to the 70-kDa form, suggesting that this second stage of processing may differ between the D and D isoforms.


Figure 5: Pulse-chase labeling of D and D receptors in transfected CHO cells. D-transfected (a) or D-transfected (b) cells were given a 15-min pulse with [S]methionine and then chased for 15 min or 1 h. The arrows indicate the 39- and 45-kDa D (a) or the 37- and 43-kDa D (b) labeled products.



Incubation of cells at 20 °C is proposed to arrest protein transport in the trans-Golgi network (TGN)(29, 30) . To test the possibility that the difference between the long and short D(2) receptor isoforms may lie in their intracellular trafficking, metabolically labeled cells were chased at 20 °C for 3 h. Fig. 6shows that the D receptor undergoes its usual processing to the mature 70-kDa species at 20 °C, whereas at this temperature, the D isoform is entirely arrested at the 45-kDa partially processed form. The fact that the D and D receptors behave differently under the same conditions of incubation may suggest that the intracellular location of this processing differs between the two receptor isoforms.


Figure 6: Effect of temperature on post-translational processing of D and D receptors. D- and D-transfected cells were given a 15-min pulse with [S]methionine at 37 °C and were then chased for 3 h at 20 or 37 °C. The arrows indicate the 45-kDa D and 70-kDa D and D products.



It is worth noting that incubation of the cells at 15 °C during the chase period, which arrests transport prior to the Golgi cisternae, did not differentiate between the subtypes and led to the accumulation of the 43-kDa D and 45-kDa D products, respectively (data not shown). Together with the equivalent rates of initial N-glycosylation shown in Fig. 3, this supports the notion that the pathways of processing of the two isoforms diverge distal to the endoplasmic reticulum (ER) where the initial rapid glycosylation occurs.

A Portion of the 45-kDa DProduct Remains Inside the Cell in Its Partially Processed Form

We have shown in Fig. 1and Fig. 4that approximately 20% of the labeled D receptor remains in the partially processed 45-kDa form after 3 h of chase. Other experiments have shown that this situation does not change for over 6 h, suggesting that this portion of the D receptor does not undergo the second stage of glycosylation. To ascertain whether this species is still located within the endoplasmic reticulum or cis-Golgi, cells expressing the D receptor were given a 15-min pulse and 3-h chase as described previously, and the immunoprecipitated products were treated with Endo H. Fig. 7shows that the 45-kDa species present after 3 h of chase is now Endo H-resistant, in contrast to the Endo H sensitivity seen in Fig. 2, where no chase was performed. Endo H cleaves only N-linked sugar moieties on proteins present in the ER and cis-Golgi. Thus the conversion from Endo H sensitivity to Endo H resistance during the 3-h chase period indicates that the 45-kDa D species has moved beyond the cis-Golgi and undergone further processing.


Figure 7: Endo H resistance of D receptor following 3 h of chase. D-expressing cells were given a pulse-chase and immunoprecipitated as described. Samples were split into two and incubated in the presence or the absence of Endo H. The arrows indicate the specific 45- and 70-kDa D products.



To determine whether the 45-kDa Endo H resistant D species has reached the cell membrane or is located inside the cell, D-expressing cells were given a pulse-chase, and prior to solubilization of cell membranes, the cells were treated with proteinase K. When added to intact cells, proteinase K will digest only extracellular domains of proteins exposed on the cell surface. Whereas the mature 70-kDa D receptor is digested by this treatment, the 45-kDa Endo H-resistant D product is protected from proteinase K digestion (Fig. 8), implying that it remains inside the cell and has not reached the cell membrane. Treatment with proteinase K in the presence of 1% Triton X-100, which solubilizes membranes, results in the digestion of all labeled proteins as expected. It therefore appears that approximately 20% of the initially labeled D receptor does not follow the same path of processing as the majority but remains inside the cell in an intracellular compartment located beyond the cis-Golgi.


Figure 8: Proteinase K treatment of D receptor in transfected CHO cells. D-transfected cells were given a pulse-chase as described and prior to solubilization were treated with 10 µg/ml proteinase K in the presence or the absence of 1% Triton X-100 for 20 min at 37 °C. The arrows indicate the specific 45- and 70-kDa D products.




DISCUSSION

In this report we have examined and compared the intracellular trafficking and glycosylation of the long and short isoforms of the D(2) dopamine receptor by investigating the manner in which carbohydrates are added to the receptor following its translation in the ER. Although the two isoforms are similarly translated and glycosylated in the ER, they display differences in their processing and intracellular trafficking in the subsequent compartments of the exocytic pathway.

The application of the pulse-chase technique to the metabolically labeled cells has enabled us to detect the newly synthesized 37- and 39-kDa receptors and to differentiate them from the 43- and 45-kDa glycosylation intermediates. The susceptibility of the latter to digestion by Endo H, the rapid rate of glycosylation, and the fact that this stage of processing occurs also at 15 °C confirm that the receptors possess N-linked carbohydrate groups added co-translationally in the endoplasmic reticulum. These 45- and 43-kDa glycosylation intermediates persist for much longer than their native protein precursors. Carbohydrate groups in general might be important in ensuring the correct charge, conformation, and stability of maturing proteins. Thus, it is possible that the initial rapid glycosylation that follows the receptor's translation occurs as a means of stabilizing the receptor following its synthesis.

The fact that D and D receptors both enter the Golgi apparatus following the initial rapid glycosylation in the ER is evident from the Endo H resistance of the remaining 45-kDa D and the 70-kDa D and D products. This is based on the fact that Endo H acts only on proteins from the endoplasmic reticulum and cis-Golgi in contrast to N-glycanase, which cleaves N-linked oligosaccharides at all stages of the glycosylation pathway.

At this point, the processing of the two isoforms appears to diverge, and although both are further glycosylated to produce a mature 70-kDa species, the nature of this glycosylation appears to differ between them. This is supported by several lines of evidence. (i) The D isoform is processed to the mature 70-kDa product faster than the D isoform. (ii) A significant portion of the D receptor remains in a post-ER intracellular compartment in its partially processed form and does not reach the plasma membrane, as evident from its resistance to both Endo H and proteinase K. (iii) At 20 °C, the D isoform is fully processed to the 70-kDa species, whereas the D isoform persists in its partially processed 45-kDa form. Incubation of cells at 20 °C is thought to arrest the transport machinery at the TGN(29, 30) , preventing trafficking from this compartment to the cell surface. Thus the D receptor is probably processed to its 70-kDa form in or proximal to the TGN, whereas the D receptor is differently processed. The effect of temperature on the D receptor may imply either that the processing occurs in an organelle distal to the TGN, which the protein does not reach due to the temperature-induced arrest of transport, or that at 20 °C the D receptor does not reach the TGN. A less likely explanation may be that the D receptor undergoes processing to the mature 70-kDa form by a different enzyme that is not fully active at 20 °C.

It is curious that the long and short isoforms should undergo differential processing in the extracellular domain where glycosylation occurs when their structural difference is located in the third intracellular domain. This is most easily explained by the idea that the two subtypes may have different conformations that cause them to become associated with different proteins involved in transport between compartments, consequently altering their post-translational processing and intracellular trafficking. It will be interesting to determine whether such differences in processing may also occur for the long and short isoforms of the D(3) receptor or other alternatively spliced members of the G protein-linked receptor superfamily.


FOOTNOTES

*
This work was supported by grants from the United States-Israel Binational Science Foundation, the Ernst and Anne Chain Research Programme, the Harry Stern Foundation, and the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-8-342618; Fax: 972-8-344141.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary cells; Endo H, endo-beta-N-acetylglucosaminidase H; TGN, trans-Golgi network; ER, endoplasmic reticulum.


ACKNOWLEDGEMENTS

We thank Dr. D. Neumann for valuable discussions.


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