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Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242
Address all correspondence and requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52240.
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Abstract |
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Introduction |
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In the course of mutagenesis studies of the rat LHR (rLHR), it has been found that many mutations of the rLHR result in an intracellular retention of the receptor (Refs. 7–14 and unpublished observations). Interestingly, as long as a region involved in hormone binding has not been disturbed, the intracellularly trapped mutant receptors bind human CG (hCG) with high affinity if the cells are first solubilized with detergent (Refs. 7–14 and unpublished observations). As such, it has been possible to assess the relative degree of intracellular retention of various LHR mutants by comparing the hCG binding activity of intact cells (representing cell surface receptors only) with the binding activity of detergent solubilized extracts (representing both cell surface as well as intracellular receptors) (9, 10, 11, 12, 13). Cells expressing the wild-type rLHR express greater than 70% of the total hCG binding activity on the cell surface (Refs. 7–14 and unpublished observations). Mutations of the rLHR have been found to have varying effects on cell surface expression. Whereas many mutants are expressed at normal levels at the cell surface (12, 15), some are trapped to moderate degrees intracellularly (9, 10, 16), and others are barely detectable at the cell surface (7, 13).
It has recently been shown that the most widely occurring mutations of the cystic fibrosis transmembrane regulator (CFTR) result in intracellular retention of this multimembrane spanning chloride channel (17). Furthermore, it has been demonstrated that incubation of cells expressing certain intracellularly retained CFTR mutants at reduced temperatures increases the percentage of CFTR protein at the cell surface (18). Because this approach has not been previously examined with mutant G protein-coupled receptors, the following studies were undertaken to examine whether reduced temperatures would similarly be an effective means of increasing the expression of mutant intracellularly retained LHRs at the plasma membrane.
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Materials and Methods |
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Temperature shift experiments
For temperature shift experiments, cells were plated onto 60-mm
dishes that had been preincubated 45 min at room temperature with 50
µg/ml fibronectin. Newly plated cells were incubated for 24 h in
growth media in a 37 C humidified incubator containing 5%
CO2. Forty-eight hours before an experiment, cells were
transferred to modulator incubator chambers filled with a humidified
mixture of 95% 02 and 5% CO2, which was then
placed in ungassed incubators at 37 C (control group), 30 C, or 26
C.
Determination of 125I-hCG binding activity at the cell
surface
125I-hCG binding assays were performed on intact
cells to determine the maximal binding capacity of cell surface
receptors (A) and on detergent soluble extracts of cells to determine
the maximal binding capacity of total (i.e. both cell
surface and intracellular) receptors (B). The percentage of hCG binding
activity on the cell surface was determined as A/B x 100.
125I-hCG binding to intact cells was performed as described
previously (9), where cells were incubated overnight at 4 C with a
saturating concentration (100 ng/ml) of 125I-hCG in the
absence or the presence of an excess of unlabeled hCG (50 IU/ml). The
preparation of detergent soluble extracts of cells and
125I-hCG binding to the soluble extracts has been
previously described (9). Aliquots of the detergent soluble extracts
were incubated overnight on ice with a saturating concentration (100
ng/ml) of 125I-hCG in the absence or the presence of an
excess of unlabeled hCG (50 IU/ml). Soluble binding assays were
completed by filtration on polyethyleneimine-treated filters (19). All
measurements were performed in duplicate.
Determination of equilibrium binding constants
Intact cells were incubated with a subsaturating concentration
of 125I-hCG in the presence of increasing concentrations of
unlabeled hCG overnight at 4 C as previously described (9). All
measurements were performed in duplicate. The data were analyzed using
the computer program LIGAND (20) to calculate the equilibrium binding
constants.
cAMP assays
For these experiments, cells were plated on fibronectin-coated
35-mm wells. After 48-h preincubations at 37 C or 26 C, the cells were
washed two times with 2 ml of warm Waymouth MB752/1 media containing 50
µg/ml gentamycin and 1 mg/ml BSA and placed in 1 ml of the same
medium containing 0.5 M isobutylmethylxanthine. After 15
min at 37 C, increasing concentrations of hCG were added and the
incubation was continued for 60 min at 37 C. The cells were then placed
on ice, and the total cAMP accumulated (in the cells and media) was
extracted by the addition of 1 ml 2 N perchloric acid
containing 360 µg/ml theophylline and then measured by RIA. All
measurements were performed in triplicate. The curve describing the
best fit of the data and the calculations of the EC50 (the
concentration of hCG required to elicit half-maximal cAMP production)
and Rmax (the maximal amount of cAMP produced) were
determined using the computer program Delta Graph.
Hormones and supplies
Highly purified hCG was kindly provided by the National Hormone
and Pituitary Agency of the NIDDK/NIH and was iodinated as described
previously (21). Modulator incubator chambers were obtained from
Billups-Rothenberg (Del Mar, CA).
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Results |
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Because intracellular forms of the wild-type rLHR or mutant rLHRs
maintain high affinity hCG binding, hCG binding assays to solubilized
cells can be used to measure the sum of intracellular plus cell surface
receptor. By comparing this with data obtained from hCG binding assays
to intact cells, which measures only cell surface receptors, it is
possible to calculate the percentage of rLHR present on the cell
surface. As shown in Table 1
, when cells expressing the
wild-type LHR were preincubated for 48 h at 37 C before the hCG
binding assays (which are performed at 4 C), 93% of the receptor was
at the cell surface. In contrast, 37 C preincubated cells expressing
either rLHR(K541, 544, 547A), rLHR(T175A), or rLHR(E441Q) expressed
only 40%, 34%, and 15% of the receptor at the cell surface,
respectively. The effects of decreased temperature on rLHR expression
were determined by comparing cells preincubated for 48 h at 37 C
with those preincubated for 48 h at 30 C or 26 C before assaying
for hormone binding. As seen in Table 1, for all the cell lines
examined, the total amount of 125I-hCG binding activity was
increased approximately 3-fold by preincubating the cells at either 30
C or 26 C, as compared with 37 C. In spite of the increase in the total
number of receptors at 30 C and 26 C, the percentage of binding
activity at the cell surface for the wild-type receptor was not
increased at the reduced temperatures. In contrast, the proportion of
125I-hCG binding activity on the cell surface for each of
the three mutants examined increased significantly at the reduced
temperatures. Thus, whereas only 40% of rLHR(K541, 544, 547A) was at
the cell surface after a 37 C preincubation, greater than 70% was on
the cell surface after 30 C or 26 C preincubations. Similarly, whereas
34% of rLHR(T175A) was on the cell surface after a 37 C preincubation,
61% and 78% were on the cell surface after 30 C and 26 C
preincubations, respectively. Although, the expression of rLHR(E441Q)
was also shifted to the cell surface, the magnitude of the shift was
not as great as with the other two mutants. Thus, rLHR(E441Q), which
expressed only 15% of its receptors at the cell surface after
preincubation at 37 C, increased its cell surface expression to 27%
and 44% after 30 C and 26 C preincubations, respectively. The data
shown in Table 1 also show that preincubations of the cells expressing
the mutant receptors at 26 C were more effective than preincubations at
30 C. In other experiments (not shown), it was determined that although
increases in cell surface expression of the mutants was apparent after
24 h at 26 C, they were not as great as those observed after a
48-h preincubation. Furthermore, a 72-h preincubation at 26 C was as
effective as a 48-h preincubation but led to a greater loss of cell
viability. Therefore, the optimal conditions for maximizing cell
surface expression without jeopardizing cell viability were determined
to be a 48-h preincubation at 26 C.
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. As can be seen from that data, within these cell lines,
only rLHR(K541, 544, 547A-c1) expressed similar numbers (albeit about
half) of cell surface receptors as rLHR(wt-18) cells. For these
reasons, the experiments assessing the functional properties of
temperature shifted mutant receptors were performed on rLHR(K541, 544,
547-c1) cells, comparing them with rLHR(wt-18) cells.
Before assessing the functional properties of rLHR(K541, 544, 547A-c1)
and rLHR(wt-18) cells after a preincubation at 26 C, we first tested
whether the binding affinity of the wild-type or mutant receptor was
altered by preincubation at reduced temperature. As shown in Table 2, intact rLHR(wt-18) cells bound hCG with a
comparable affinity after a 48-h preincubation at 26 C as compared with
cells preincubated at 37 C. Similarly, intact rLHR(K541, 544, 547A-c1)
cells bound hCG with the same high affinity after a 26 C preincubation
as after a 37 C preincubation. We next examined the ability of each of
the cell lines to respond to hCG with increased cAMP after
preincubations at 37 C vs. 26 C. After preincubating each
group of cells for 48 h at either 37 C or 26 C, all cells were
then incubated at 37 C in the absence of hormone or with increasing
concentrations of hCG. As summarized in Table 3, when
the cells were preincubated at 37 C, the Rmax of rLHR(K541,
544, 547A-c1) cells somewhat greater than that of rLHR(wt-18) cells and
the EC50 values of the two cell lines were comparable.
After a 48-h preincubation at 26 C, the Rmax and
EC50 values for both the rLHR(K541, 544, 547A-c1) and
rLHR(wt-18) cells were comparable with the same cell line preincubated
at 37 C. These data suggest that at these receptor densities the
Rmax is not proportional to cell surface receptor numbers.
This conclusion is further supported by earlier studies comparing the
responsiveness of rLHR(wt-18) cells as compared with a cell line
expressing 4-fold higher numbers of cell surface wild-type rLHRs (11)
and is consistent with the model put forth by Whaley et al.
describing the relationship between Rmax and cell surface
receptor numbers (22). The observation, however, that the
responsiveness of the rLHR(K541, 544, 547A-c1) cells after the 26 C
preincubation is as good, if not better, as compared with the
rLHR(wt-18) cells after the 26 C preincubation suggests that the
increased cell surface mutant receptors expressed after a 48-h
preincubation of the cells at 26 C are capable of transducing the
signal of hormone binding.
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. As shown in Fig. 1, a 48-h preincubation at 26 C
resulted in 75% of the rLHR(T175A) being on the cell surface as
opposed to only 19% if the cells were preincubated for the same length
of time at 37 C. For control purposes, cells were also preincubated for
72 h at 26 C vs. 37 C. The same trend was apparent,
where 100% of the mutant was on the cell surface after 3 days at 26 C
vs. 20% of the mutant after 3 days at 37 C. To examine the
time-course of reversibility, cells were preincubated at 26 C for
48 h and then shifted to 37 C for 2 h, 6 h, or 24
h. As shown in Fig. 1, the percentage of mutant receptor at the cell
surface remained elevated even after 6 h of being returned to 37
C. However, by 24 h after being shifted back to 37 C, the
percentage of mutant receptor at the cell surface was decreasing.
Nonetheless, it was still elevated compared with cells having been at
37 C continuously. Similar results were observed for rLHR(K541, 544,
547A-c1) cells in that cells preincubated for 48 h at 26 C and
then 6 h at 37 C showed a comparable percentage of cell surface
receptors as cells preincubated for 48 h at 26 C only (data not
shown). Taken altogether, these data show that although the effects of
reduced temperature on cell surface expression of LHR mutants are
reversible, the time course is slow enough to allow for most assays of
receptor function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F508), (17) is
temperature sensitive (18). Thus, it is detected on the cell surface in
a functional form when expressed in Xenopus oocytes or Sf9 insect
cells, which are typically grown at temperatures less than 37 C, but
not in mammalian cells maintained at 37 C. However, if mammalian cells
expressing the CFTR(F508) mutant are preincubated for 2 days at
reduced temperatures, then functionally active CFTR protein is
expressed at the cell surface (18). Because many mutations of the rLHR
introduced by site-directed mutagenesis have also been shown to result
in intracellular retention (Refs. 7–14 and unpublished observations),
we thought it important to test whether a multimembrane spanning G
protein-coupled receptor mutant might also be temperature
sensitive.
To examine this question, three mutants LHRs were examined. Each of
these mutants has been shown to be retained to some degree
intracellularly, indicating that the mutations caused some degree of
misfolding into the proteins. Nonetheless, as has been shown for other
rLHR mutants where a binding site has not been disturbed, these
intracellularly trapped mutants exhibited high affinity hCG binding,
suggesting that the misfolding was not a gross alteration of
conformation (9, 10, 12). Furthermore, the intracellular retention of
these mutants does not appear to be a result of overexpression of the
mutant proteins resulting in overloading the cellular processing
system. As shown in Table 1, the cell lines expressing the mutants all
had lower numbers of total rLHRs than rLHR(wt-18), which has a density
of approximately 3,000–6,000 cell surface receptors per cell (9, 11, 24). As such, all the cell lines in this study expressed receptors at
densities somewhat lower than gonadal cells expressing wild-type LHRs.
Furthermore, for both the wild-type rLHR as well as rLHR(T175) and
rLHR(K541, 544, 547) several different clones of cells were examined.
For each kind of receptor, the absolute numbers of cell surface
receptors varied greatly between clones; however, the percentage of
receptor at the cell surface was similar. Therefore, the intracellular
retention does not appear to be dependent upon receptor density. The
experiments presented herein demonstrate that the rLHR mutants
rLHR(T175A), rLHR(K541, 544, 547A), and rLHR(E441Q) are indeed
temperature sensitive in that a greater percentage of receptor can be
expressed on the cell surface if the cells are grown at reduced
temperatures. In other studies, we have found that not all rLHR mutants
that are trapped intracellularly are temperature sensitive (13). In
general, it has been observed that if a mutant is profoundly trapped
such that little or no cell surface binding activity is detected, then
it is unlikely that reduced temperatures will shift a greater
proportion of the mutant to the cell surface. For example, when
deletions were made in portions of the extracellular loops of the rLHR,
most of these mutants were expressed almost entirely intracellularly
(where they exhibited high affinity hCG binding) and were not shifted
to the cell surface after preincubations at 26 C (13). In another case,
deletions were made of the individual leucine-repeats of the
extracellular domain of the rLHR. Deletions of repeats 1, 2, 3, 4, 5,
or 6 did not cause a loss of expression of receptor protein but did
result in a complete loss of hormone binding activity in both intact
and solubilized cells, suggesting that these deletions disrupted a
hormone binding domain(s) (7). Preincubations of cells expressing any
of these mutants did not cause binding activity to appear in either the
intact or solubilized cells (unpublished data), presumably due to the
conformational changes caused by such large deletions. In contrast to
the above situations, if rLHR mutants are expressed to at least some
degree at the cell surface at 37 C, then they generally do respond at
least somewhat to the decreased temperatures. This trend is apparent
within the three mutants examined herein in that rLHR(E441Q), which was
trapped more extensively than the other two mutants, responded less
well to the reduced temperatures. Therefore, we postulate that a
decreased percentage of mutant receptor on the cell surface coupled
with an inability to increase cell surface expression after
preincubations at decreased temperatures is most likely indicative of
more severe conformational changes imposed by the particular
mutation.
The data presented herein demonstrate that preincubations of cells
expressing either the wild-type or intracellularly retained mutant
rLHRs at reduced temperatures causes an increase in the total number of
rLHRs. Furthermore, for cells expressing intracellularly retained
mutant rLHRs, the decreased temperatures increase the percentage of
receptor at the cell surface. Thus, the overall efficiency of
expression of a misfolded receptor is increased. At which stage of the
folding/trafficking process the reduced temperatures have an effect has
not yet been determined. Nor is it known how reduced temperatures aid
in the folding of otherwise misfolded proteins. However, some important
insights may be extrapolated from recent studies on the thermodynamic
properties of CFTR(F508), which suggest that the mutation affects a
step in the folding pathway rather than the protein’s stability (25).
Hence, we speculate that those mutations of the rLHR that are
temperature sensitive may similarly have affected residues that make
important intramolecular contacts during the folding process of the
molecule.
Mutants of the rLHR that are not expressed at the cell surface appear to be retained in the endoplasmic reticulum (ER) and/or cis-Golgi because they are sensitive to endoglycosidase H (14, 16). Because intracellularly retained rLHR mutants have been found to have a high affinity for hCG, it appears that by the time they exit the ER/cis-Golgi, they must have already assumed most if not all of their final conformation. Recent studies, however, have shown that the intracellularly retained rLHR mutants, like the precursor form of the wild-type rLHR, are not yet in the same conformation as the cell surface mature form of the receptor (13). This conclusion is based upon the observation that oLH, unlike hCG, binds the intracellular form of the rLHR with a reduced affinity as compared with the cell surface rLHR (13). Therefore, modifications to the tertiary structure of the rLHR must ensue after its exit from the ER/cis-Golgi that allow it to have the proper final conformation enabling plasma membrane insertion.
Mutations of G protein-coupled receptors that result in intracellular retention are not restricted to the rLHR. Indeed, when the same mutations causing intracellular retention of the rLHR have been introduced into the rFSHR, it has been shown that the rFSHR mutants are similarly retained intracellularly (14). However, unlike the rLHR, intracellularly retained mutants of the rFSHR are devoid of any hormone binding activity (14). The presence of high affinity hCG binding activity (with albeit decreased oLH binding activity) in intracellularly retained rLHR mutants, but the absence of any hormone binding activity in intracellular retained rFSHR mutants suggests a major difference in the temporal pattern of folding between the two gonadotropin receptors during their biosynthesis. Consistent with this hypothesis is the observation that whereas the newly synthesized rFSHR absolutely requires N-linked carbohydrates to fold into a conformation enabling the receptor to bind hormone (26), the rLHR does not strictly require N-linked carbohydrates for folding into its mature conformation (D. Davis and D. L. Segaloff, manuscript submitted). An interesting question is whether mutations of the rLHR and rFSHR, which cause intracellular retention, also cause intracellular retention of the closely related TSHR. Certainly, many mutants of the hTSHR have been shown to result in the absence of hormone binding activity on the cell surface (27, 28, 29). However, in the absence of binding assays to solubilized extracts or Western blots, it is not possible to conclude whether the absence of binding activity on the cell surface is due to intracellular retention of the mutants and/or decreased stability of the mutants. Similarly, certain mutations of several other G protein-coupled receptors have been shown to result in a decreased cell surface expression of the mutant receptor. Again, whether these result from decreased protein expression and/or decreased cell surface expression needs to be determined. It has, however, been clearly shown for adrenergic receptors (30) and rhodopsin (31) that some mutations do indeed result in intracellular retention of these G protein-coupled receptors.
As discussed above, cystic fibrosis is associated with mutations of the CFTR protein that prevent its normal cell surface expression (17). Other diseases, though, have also been associated with intracellular trapping of mutant cell surface receptors. For example, autosomal dominant retinitis pigmentosa has been shown to be associated in many cases with mutations causing single amino acid substitutions in rhodopsin that resulted in partial or extensive trapping of the mutants within the endoplasmic reticulum (31). Similarly, a large percentage of the mutations of the LDL receptor associated with familial hypercholesterolemia resulted in the intracellular retention of the mutant LDL receptors (32). Of particular relevance is the report of a naturally occurring mutations of the hLHR found in a patient with Leydig cell hypoplasia (33). In this case, the decreased cell surface expression of a LHR prematurely truncated in the fifth transmembrane helix appeared to be due at least in part to intracellular retention of the mutant receptor (33). It is not unreasonable to expect that many other naturally occurring mutations of the LHR would also lead to intracellular retention of the receptor and consequent impairment of reproductive function. For those mutations that are not affecting hormone binding or G protein-coupling per se, a therapeutic intervention that would increase the proper folding and targeting of the receptors to the plasma membrane would be predicted to be sufficient for conferring responsiveness of the gonadal cell. As such, it will be important to more fully understand the normal processes of folding, posttranslational processing, and cell surface targeting of the LHR.
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Acknowledgments |
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Footnotes |
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2 Recipient of NIH Research Career Development Award HD-00968.
Received July 18, 1996.
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References |
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