(Received for publication, August 1, 1994; and in revised form, September 8, 1994)
From the
The present studies were done to evaluate the role of the
cytoplasmic tail of the G-protein-coupled receptor for parathyroid
hormone (PTH) and PTH-related protein (PTHrP) in the endocytosis of
agonist-occupied receptors. PTH/PTHrP receptor mutants progressively
truncated from the C terminus were expressed in COS-7 cells, and their
ability to internalize I-PTHrP(1-34)amide was
determined. Most of the C-terminal tail (91 of 127 residues) could be
deleted without affecting internalization. However, further truncation
removing residues 475-494 resulted in a 50-60% decrease in
ligand internalization. A mutant with an internal deletion of these 20
amino acids showed a similar reduction in internalization, confirming
the presence of a positive endocytic signal. No additional positive
signals were found in the membrane-proximal region of the tail.
However, alanine mutagenesis of the membrane-proximal residues
459-461 (EVQ
AAA) resulted in a mutant PTH/PTHrP receptor
displaying a 40% increase in ligand endocytosis, indicating that EVQ
functions as a negative signal. Treatment of COS-7 cells with
hypertonic sucrose (to disrupt clathrin lattices) markedly suppressed
(by >80%) PTH/PTHrP receptor internalization. These results
demonstrate the presence of both positive and negative endocytic
signals in the membrane-proximal cytoplasmic tail of the PTH/PTHrP
receptor and suggest that these signals regulate the ability of the
receptor to accumulate in clathrin-coated pits.
Endocytosis of cell surface receptors is a ubiquitous process that serves multiple functions(1, 2, 3) . Constitutively recycling receptors, such as those for transferrin and low density lipoprotein, are shuttles that deliver their ligands to the cell interior and then return to the plasma membrane. Signaling receptors, on the other hand, are generally not endocytosed efficiently until agonist is bound and transmembrane signaling has occurred. At this point, endocytosis may allow receptor resensitization (e.g. by targeting the receptor to an acidic endosomal compartment that facilitates ligand dissociation, followed by receptor recycling) or may serve as the initial step in the ultimate lysosomal degradation of the receptor (down-regulation)(4) .
For many receptors,
efficient endocytosis requires discrete cytoplasmic receptor
determinants that serve as signals for interaction with the endocytic
machinery of the cell. Positive internalization signals have been
identified in constitutively recycling receptors such as transferrin
receptors, low density lipoprotein receptors, cation-independent
mannose 6-phosphate receptor, and asialoglycoprotein
receptors(5, 6) . They are composed of a short linear
stretch of cytoplasmic amino acid residues which lack an obvious
consensus sequence, but share certain common features. These motifs
generally consist of 6 residues that are 20-50 amino acid
residues from the plasma membrane and frequently contain an essential
tyrosine residue (7) . Such sequences are thought to be
directly involved in the association of the receptor with the HA-2
adapter proteins of the membrane clathrin lattice, a process that leads
to efficient endocytosis via clathrin-coated pits(8) .
Relatively little is known concerning the molecular basis of
endocytosis of heptahelical G-protein-coupled receptors (GPCRs). ()Clathrin-coated pits/vesicles have been implicated as
mediating internalization of some(9) , but not
other(10, 11) , GPCRs. The sequence NPXXY,
similar to the NPXY internalization motif in the low density
lipoprotein receptor(5) , is required for efficient
agonist-mediated sequestration of the
-adrenergic receptor away
from the cell surface(12) . This sequence is highly conserved
in the seventh membrane-spanning segment of many GPCRs and might serve
as a common endocytic signal. Serine/threonine-rich sequences in the
third cytoplasmic loop or cytoplasmic tail contribute to efficient
internalization of the GPCRs for thyrotropin-releasing
hormone(13) , gastrin-releasing peptide(14) ,
muscarinic agonists(15) , and yeast
-factor(16) .
The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) (17) is a member of a newly recognized, structurally distinct heptahelical GPCR subfamily that also includes receptors for secretin, glucagon and related peptides, calcitonin, and several others(18) . Members of this subfamily lack the conserved NPXXY sequence; nonetheless, they undergo efficient ligand-mediated endocytosis. The sequence elements in these receptors required for this process have not been identified. Previous studies of the PTH/PTHrP receptor suggest that endocytosis leading to down-regulation of the receptor occurs in bone and kidney cells in vitro(19, 20) and may serve to regulate target cell responsiveness in vivo(21) .
In the present study, we have used a mutagenesis strategy to identify receptor sequences in the cytoplasmic tail of the PTH/PTHrP receptor that influence receptor-mediated endocytosis. The results indicate the presence of two novel determinants. One is within residues 475-494 and is required for efficient receptor internalization; the second is within residues 459-461, which are highly conserved in the PTH/secretin receptor subfamily, and serves to suppress internalization. These studies illustrate that at least one member of the PTH/secretin receptor subfamily of GPCRs is endocytosed by a signal-dependent mechanism and that such signals can suppress or enhance internalization.
Internal deletion and tandem alanine mutation constructs were generated by overlap extension PCR, as described by Pease and co-workers(22) . For each receptor construct, four oligonucleotide primers were utilized, two internal primers bearing desired mutation sequences encoding tandem alanines or single alanine and two external primers bearing the 5` end sequences of the final PCR fragment. Primers were designed such that their melting temperatures are between 55 °C and 65 °C. Two overlapping fragments gel-purified from two PCR reactions with paired external and internal primers were mixed for a second PCR reaction. At a low but significant frequency, two overlapping strands were annealed and extended by the DNA polymerase to generate a full-length template which was then amplified by two external primers. The external primer sites are chosen such that the PCR fragment includes a NsiI and SphI sites to facilitate subcloning into the plasmid of interest. The PCR fragments were gel-purified, digested with SphI and NsiI, and ligated with the predigested OKO2 vector. All receptor constructs derived from pcDNA1/AMP (Invitrogen Corp.) were transformed into, maintained, and amplified in E. coli strain, Top10F`. Plasmid DNAs were purified using QIAGEN columns according to the manufacturer's recommended procedure (QIAGEN Corp.). The PCR portions of the final constructs were sequenced to confirm the identity of the PCR product.
Fig. 1presents a diagrammatic representation of the mutant OK PTH/PTHrP receptors encoded by the constructs used in this study. These include a series of C-terminal truncation mutants, designated by ``T'' followed by the residue number of the last amino acid. Also shown are 3 mutants (``D'') in which the residues shown were deleted and replaced by a set of 4 alanine residues.
Figure 1: A schematic diagram of wild type and mutant PTH/PTHrP receptors evaluated in the present study. Progressive truncation at the cytoplasmic tail resulted in mutant PTH/PTHrP receptors (T507, T494, T474, T466, and T458) containing 49, 36, 16, 8, and 0 amino acid residue tails, respectively. In mutant receptors D459-462, D463-466, and D475-494, cytoplasmic tail residues (1-4, 5-8, and 17-36, respectively) were replaced by 4 alanines.
Functional expression of wild-type OK PTH/PTHrP receptors
was assessed in COS-7 cells transiently transfected with expression
vector OKO2 (Fig. 2). These cells have no detectable endogenous
PTH/PTHrP receptors and are thus a suitable model system for these
studies. Competitive binding studies (Fig. 2A)
demonstrated the presence of PTH/PTHrP receptors that display an
apparent affinity (IC) of 4.5 nM, similar to the
reported value in an opossum kidney cell line(26) . These
receptors were found to be functionally coupled to adenylyl cyclase (Fig. 2B), conferring PTH-dependent increases in cAMP
levels, with an EC
for bPTH(1-34) of 0.21
nM.
Figure 2:
I-labeled
PTHrP(1-34)amide binding (A) and
bPTH(1-34)-stimulated cAMP production (B) in COS-7 cells
transiently expressing the wild-type OK PTH/PTHrP receptor. Binding and
cAMP data represent the mean ± S.E. of 8 experiments. Total
specific binding was 27 ± 3% of added tracer, and the IC
for bPTH(1-34) was 4.5 ± 0.7 nM. Basal and
maximal bPTH(1-34)-stimulated cAMP levels were 8 ± 2 and
75 ± 14 (pmol/30-mm well), and the EC
for
bPTH(1-34) was 0.21 ± 0.03
nM.
Fig. 3compares the kinetics of ligand binding and of receptor internalization in COS-7 cells expressing either wild-type PTH/PTHrP receptors or receptors lacking all but 16 residues in the cytoplasmic tail (T474). These receptors displayed similar ligand binding kinetics (Fig. 3A) and binding affinity (data not shown), but differed markedly in their internalization properties (Fig. 3B). Internalization of the wild-type receptor was rapid (half-maximal within 5 min) and reached a maximal level of 50-60% of cell-associated ligand within 20-30 min. Truncation mutant T474 displayed a similar internalization time course, but only about 25% of cell-associated ligand was internalized after 60 min. To define in more detail the determinants in the cytoplasmic tail required for efficient receptor endocytosis, we evaluated a series of PTH/PTHrP receptor mutants progressively truncated at the C terminus of the cytoplasmic tail (Fig. 4). Functional expression of these mutant receptors, assessed by radioligand binding (Fig. 4A), was variably reduced compared to wild-type for all mutants except T474. No significant differences in binding affinity were seen (data not shown). Results of internalization assays (Fig. 4B) revealed that T507 and T494 were internalized indistinguishably from wild-type, whereas both T474 (as shown above) and T466 were internalized with a 50-60% reduced efficiency.
Figure 3:
Time course of PTH/PTHrP receptor
internalization in COS-7 cells. The wild-type receptor and mutant T474
were transiently expressed in COS-7 cells, and I-PTHrP(1-34)amide binding (A) and
internalization (B) were assessed, as described under
``Experimental Procedures.'' Similar results were obtained in
two experiments, each with triplicate data
points.
Figure 4: Effects of C-terminal truncation and internal deletion on PTH/PTHrP receptor binding (A) and endocytosis (B). Mutant receptors are as described in Fig. 1. Values are normalized to those of the wild-type receptor which was assigned a value of 100%. Data represent the mean ± S.E. of three to five experiments with triplicate in each. Values with asterisks are significantly different from wild-type (p < 0.005, Student's t test).
These results with truncation mutants suggest the presence within amino acid residues 475-494 of a determinant of efficient PTH/PTHrP receptor endocytosis. However, it is also possible that reduced internalization was due to local effects such as the change in the relative position of the C terminus in T474 and T466. Moreover, as redundant endocytic codes/regions have been identified in a number of receptors(13, 27) , it is also possible that reduced internalization required the deletion of more C-terminal sequence as well as residues 475-494. To investigate these possibilities, an internal deletion mutant (D475-494) in which a sequence region between amino acid residues 475 and 494 was replaced by 4 alanine residues was generated and expressed in COS-7 cells. As shown in Fig. 4B, internalization of this mutant receptor was reduced by 50%, similar to the defect seen with truncation mutant T474. These results indicate that residues 475-494 contain a signal that is required for efficient PTH/PTHrP receptor internalization, and that this signal is functional in the context of the full-length PTH/PTHrP receptor.
Because the truncation mutant T458 was not expressed on the cell surface, it was unclear whether juxtamembrane residues 459-466 contributed to receptor internalization. To investigate this, we generated two tandem alanine mutations targeting this region. The two mutated receptors (D459-462 and D463-466) were expressed at levels somewhat lower than wild-type, based on their ligand binding properties (Fig. 5A). Strikingly, D459-462 displayed a 40% increase in internalization relative to wild-type. D463-466 did not differ from wild-type. These results suggest that the juxtamembrane region of the cytoplasmic tail contains a second endocytic signal that serves to constrain receptor internalization.
Figure 5: Mutations in the juxtamembrane region of the cytoplasmic tail of the PTH/PTHrP receptor: effects on receptor binding (A) and endocytosis (B). Mutant receptors are as described in Fig. 1. Data represent the mean ± S.E. of three experiments with triplicate samples in each. Values with asterisks are significantly different from wild-type (p < 0.005, Student's t test)
Exposure of cells to hypertonic medium has
been shown to remove membrane-associated clathrin lattices, resulting
in a loss of clathrin-coated pits and vesicles(28) . When COS-7
cells transiently expressing the wild-type OK PTH/PTHrP receptor were
exposed to hypertonic solution (0.45 M sucrose) 1 h before the
addition of I-PTHrP(1-34)amide, there was an 82%
reduction in receptor internalization with no effect on receptor
binding per se (Fig. 6). This effect was fully
reversible upon restoration of conditions that allow clathrin
reassembly (removal of the sucrose). These results are consistent with
the notion that ligand-induced PTH/PTHrP receptor internalization
occurs via clathrin-coated pits. The internalization of the mutant
T474, which is deficient under standard incubation conditions, is
further reduced by hypertonic sucrose, indicating that clathrin
pit-mediated internalization of the PTH/PTHrP receptor is not solely
dependent upon the positive endocytic signal in the tail.
Figure 6:
Effect of hypertonic sucrose treatment on
PTH/PTHrP receptor binding (A) and endocytosis (B).
COS-7 cells transiently expressing the wild-type or the T474 mutant
PTH/PTHrP receptor were preincubated ± hypertonic media (0.45 M sucrose) for 1 h, followed by a 1-h incubation with I-PTHrP(1-34) ± 0.45 M sucrose, as
indicated. Receptor binding and internalization were then assessed as
described under ``Experimental Procedures.'' Values are
normalized to those of the wild-type receptor which was assigned a
value of 100%. Data represent the mean of two to three independent
experiments with triplicate samples in
each.
Previous studies have suggested that homologous down-regulation of the PTH/PTHrP receptor may limit target cell responsiveness under conditions of ligand excess in vivo(21) , and that this process is associated with receptor internalization(19, 20, 29) . Molecular cloning of the PTH/PTHrP receptor (17) has now allowed us to address the molecular basis of receptor-mediated endocytosis. The results of the present study indicate that the 90 C-terminal amino acid residues of the cytoplasmic tail can be removed without affecting PTH/PTHrP receptor-mediated endocytosis. However, the proximal region of the tail contains two discrete sequence elements that influence internalization, one a positive endocytic signal between residues 17 and 36 of the tail, and the other a negative signal comprising 3 residues at the interface between membrane and cytoplasm.
Two approaches were used to identify the positive endocytic signal. Expression of C-terminal truncation mutants revealed that wild-type levels of receptor-mediated endocytosis were obtained with PTH/PTHrP receptors possessing only a 36amino acid tail. However, further truncation deleting residues LDFKRKARSGSSTYSYGPMV resulted in markedly (50-60%) reduced receptor internalization. Therefore, in the context of a severely truncated PTH/PTHrP receptor, this sequence is sufficient to signal enhanced endocytic efficiency. Moreover, this sequence is required for efficient endocytosis since a nontruncated receptor in which this sequence was specifically deleted displayed comparably reduced endocytosis.
The relationship between the signal
contained within this sequence and previously identified positive
endocytic signals is unclear. The sequence does not contain a
heretofore identified internalization signal, but resembles known
signals in certain respects. It is rich in serine/threonine residues as
are internalization sequences in the cytoplasmic tails of the
thyrotropin-releasing hormone(13) , gastrin-releasing
peptide(14) , and yeast -factor (16) receptors.
Recently, we have found that the cytoplasmic tail of the PTH/PTHrP
receptor is rapidly phosphorylated upon agonist binding, (
)a
modification that could promote receptor internalization. Also present
are 2 tyrosine residues in a stretch of sequence likely to have
-turn structure by Chou-Fasman analysis(30) . A tyrosine
residue in the context of a
-turn is a characteristic feature of
internalization signals in several constitutively recycling receptors (7) and could play a similar role in signaling receptors. In
this regard, preliminary mutagenesis studies indicate that the positive
signal is contained within the sequence TYSYGPMV. (
)This
stretch contains 2 tyrosines included in the sequence TYSYG which has a
high probability of forming a turn(30) . Unlike most of the
cytoplasmic tail, this sequence is highly conserved among PTH/PTHrP
receptors from opossum to human(18) , suggesting a functional
role. Further mutagenesis should serve to clarify the role of
serine/threonine phosphorylation and/or tyrosine residues in the
endocytic function of this domain.
Mutated PTH/PTHrP receptors lacking the positive internalization signal (e.g. T474) are endocytosed, albeit to a reduced extent. This is not due entirely to bulk flow internalization, as disruption of clathrin lattices by treatment with hypertonic sucrose further suppressed internalization. The present signal may act in concert with one or more additional positive endocytic signals located outside of the receptor's cytoplasmic tail. It will be important to establish whether this signal can function autonomously to promote endocytosis, e.g. when spliced onto an internalization-defective transferrin receptor.
An
important issue for signaling receptors is the relationship between
signal transduction and receptor internalization. In the case of
epidermal growth factor and insulin receptors, mutations that abolish
intrinsic tyrosine kinase activity markedly reduce receptor-mediated
endocytosis(31, 32) . In the case of the epidermal
growth factor receptor, tyrosine kinase activity, but not
autophosphorylation, is required, indicating that signal transduction
and heterologous protein phosphorylation is a prerequisite for
efficient internalization(32, 33) . For GPCRs,
endocytic signals may be found in cytoplasmic domains that are either
independent of (34) or overlap with (35) regions that
are required for agonist-stimulated signal transduction. In the present
study, truncation mutants lacking the positive endocytic signal appear
to function similarly to wild-type with respect to adenylyl cyclase
activation, indicating that internalization can be
disrupted in mutants that retain the ability to signal adenylyl
cyclase. We do not yet know whether the results of transmembrane
signaling (e.g. activation of protein kinase A or protein
kinase C) are involved in regulating PTH/PTHrP receptor endocytosis.
Tandem alanine mutagenesis of the proximal region of the cytoplasmic tail revealed a second domain that influences PTH/PTHrP receptor-mediated endocytosis. Mutation D459-461, in which residues EVQ at the membrane-cytoplasmic interface were mutated to AAA, displayed a marked, 40% increase in internalization. This result suggests that the EVQ sequence serves to limit the entry of receptors into endocytosis-competent structures, either by facilitating interaction of the receptor with poorly endocytosed membrane components or by directly inhibiting the function of a positive endocytic signal(s). It is noteworthy that this EVQ sequence is highly conserved across members of the PTH/secretin receptor subfamily (but not in other GPCRs) and may serve as a suppressor of endocytosis in other subfamily members. Although the vast majority of endocytic signals previously described are positive signals, a juxtamembrane cytoplasmic domain that inhibits coated-pit localization and endocytosis has been identified in an isoform of the Fc receptor(36) . In that case, the minimum sequence required for inhibition is not yet clear. Rodriguez et al.(37) found that truncation of the distal portion of the cytoplasmic tail of the luteinizing hormone/human choriogonadotropin receptor resulted in enhanced receptor-mediated hormone internalization, suggesting the removal of a negative endocytic signal. Thus, these results and ours indicate that receptor-mediated endocytosis is a highly complex and regulated process, with multiple receptor domains playing facilitory as well as inhibitory roles.
Both clathrin-coated and noncoated endocytic pathways have been
suggested for GPCRs. For example, -adrenergic receptors have been
reported to be internalized through noncoated vesicles(11) .
However, a recent report showed that
-adrenergic receptors
co-localized in endosomes with transferrin, which is known to be
internalized in clathrin-coated pits/vesicles(38) . It is not
clear whether this discrepancy is due to differences in the cell lines
used or whether noncoated vesicles are able to fuse with
clathrin-coated vesicles. The marked inhibition of PTH/PTHrP receptor
internalization produced by hypertonic sucrose, a treatment known to
disrupt clathrin lattices (28) , suggests that PTH/PTHrP
receptor endocytosis occurs at least in part through a clathrin-coated
pit mechanism. This result is consistent with the previous report of
Silve et al.(39) who used EM autoradiography to
demonstrate the presence of iodinated PTH in clathrin-coated pits on
the surface of cells in calvarial bone.
In summary, specific positive and negative signals in the cytoplasmic tail of a GPCR, the PTH/PTHrP receptor, serve to regulate agonist-stimulated endocytosis via clathrin-coated pits/vesicles. These signals are distinct from those previously identified in both constitutively recycling and signaling receptors. Further delineation of the critical features of these signals and identification of their targets should provide new insights into the molecular basis of endocytosis of the PTH/PTHrP receptor and possibly other GPCRs.