(Received for publication, July 13, 1995; and in revised form, September 25, 1995)
From the
The D 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
receptor may lie in their post-translational processing and
intracellular trafficking.
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
, D
, and
D
)(7, 8, 9) . This heterogeneity
was found to be yet further diverse by the identification of two
alternatively spliced isoforms of the D
(10, 11, 12) and subsequently D
(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
) or 21 (D
) 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 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
receptor isoforms may differ in their
post-translational modifications. The fact that D
-type
dopamine receptors are glycosylated was established by early
photoaffinity labeling studies that utilized glycosidase treatment to
demonstrate the presence of oligosaccharides on D
-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 ()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
receptor and present evidence
that the D
and D
receptors are differently
glycosylated and are subject to different patterns of intracellular
trafficking.
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
-specific antibody in the presence or absence of
inhibiting D
-peptide or with D
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
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
receptors. In addition, the difference between the long and short
D
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
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
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
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
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.
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 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.
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.
In this report we have examined and compared the
intracellular trafficking and glycosylation of the long and short
isoforms of the D 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 receptor or other alternatively spliced
members of the G protein-linked receptor superfamily.