(Received for publication, October 27, 1994; and in revised form, November 29, 1994)
From the
In order to identify the amino acid sequences responsible for the internalization of the cloned rat brain neurotensin receptor, we carried out site-directed mutagenesis of the cDNA encoding the receptor followed by expression of the receptor into mammalian COS 7 cells. In cells transfected with the full-length neurotensin receptor, 56% of iodinated neurotensin specifically bound to the cells after 60 min of incubation at 37 °C was internalized. Deletions made in the third intracellular loop did not affect receptor internalization. By contrast, internalization was reduced to 5% of total in cells in which almost all the carboxyl-terminal tail of the receptor had been deleted (R392stop). In order to determine which part of the tail was responsible for this effect, several Ser and Thr residues were deleted in the carboxyl cytoplasmic sequence of the receptor. Almost all of these receptors were internalized as efficiently as the wild type. Only the form of the neurotensin receptor truncated at Glu-421 (deletion of the last three residues, TLY) produced a significant decrease in the amount of ligand internalized. Finally, point mutations of Thr-422 and Tyr-424 residues to Gly led to an almost complete loss of ligand internalization demonstrating the involvement of these 2 residues in the internalization process. Replacement of the last three amino acids by the cytoplasmic endocytosis signal of the vesicular stomatitis virus did not restore the efficiency of neurotensin receptor internalization. These biochemical results were confirmed by confocal microscopic analysis. Cells transfected with the wild type receptor showed a temperaturedependent intracellular accumulation of a fluorescent analog of neurotensin, whereas cells transfected with a receptor truncated at the carboxyl terminus showed a clustering of the fluorescent peptide at the cell surface.
Plasma membrane neurotensin receptors (NTRs) ()have
been described as being rapidly sequestrated from the cell surface
after interaction with their ligand(1, 2) . This
sequestration process is followed by a rapid internalization of
receptor-ligand complexes, which is both time- and
temperature-dependent(2, 3) . The region of the
receptor responsible for the internalization process has not yet been
defined. However, studies on the internalization of other
G-protein-coupled receptors (GPCRs) have singled out two intracellular
domains essential for ligand-induced internalization: the third
cytoplasmic loop in the case of the muscarinic cholinergic receptor (4) and the carboxyl terminus in the case of both the
thyrotropin-releasing hormone (5) and gastrin-releasing peptide
receptors(6) . In all three cases, Ser and Thr residues were
found to be critical for receptor sequestration suggesting that they
may be critical for internalization of all G-protein-coupled receptors.
The two putative palmitoylation sites, Cys-X-Cys, in the
cytoplasmic tail of the thyrotropin-releasing hormone receptor were
also shown to be involved in the internalization process(5) .
More recently, the highly conserved Tyr residue in the NPXXY
sequence present in the seventh transmembrane domain of almost all
GPCRs has been shown to be required for agonist-mediated
internalization of the
-adrenergic receptor (7) but not for that of the gastrin-releasing peptide
receptor(8) .
The only rat neurotensin receptor cloned to date (9) possesses all of the amino acid sequences implicated in the internalization of other GPCRs; the third cytoplasmic loop contains several Ser and Thr residues, while the intracellular tail bears four clusters of Ser and Thr and a Cys-X-Cys(386-388) sequence. We have therefore studied the influence of these different regions on neurotensin-induced receptor internalization using site-directed mutagenesis and expression of the mutated receptors into mammalian cells.
Figure 1:
Topographical model of the different
point mutations and deletions of the carboxyl-terminal region of the
neurotensin receptor. The final 52 amino acids (residues 373-424)
of the wild type rat neurotensin receptor are depicted. TM7 refers to the end of the seventh transmembrane domain, and the asterisk indicates the carboxyl terminus. X indicates
a deleted amino acid. G represents a point substitution with a
Gly residue. Italicletters refer to the tagged
sequence added after Glu-421 for the 422-424-VSV mutant. All
mutants were created by site-directed
mutagenesis.
Internalization experiments were performed by suspending
dissociated cells (0.5 10
/ml) in an
Earle-HEPES-Tris 25 mM buffer, pH 7.5, supplemented with 0.1%
glucose and 0.1% bovine serum albumin, containing 0.8 mM 1,10-phenanthroline and 0.1 µMN-benzyloxycarbonyl-prolylprolinal to prevent ligand
degradation. Suspended cells (200 µl) were incubated at 37 °C
with 0.2 nM
-
I-BH-NT(2-13) for
various periods of time in a total volume of 250 µl in the presence
or in the absence of 10 µM phenylarsine oxide, an
internalization blocker. At the end of the incubation, 25 µl of a 1 M acetic acid solution containing 5 M NaCl was added
in samples for 2 min at 37 °C to dissociate surface-bound ligand
molecules. Cells were then filtered through GF/C glass fiber filters
(Millipore), and filters were washed twice with 5 ml of ice-cold buffer
before counting. In all cases, parallel incubations were conducted in
the presence of 1 µM unlabeled NT to determine nonspecific
binding. The efficiency of internalization was expressed as the percent
of specific
-
I-BH-NT(2-13) binding that was
resistant to the acid-NaCl wash(3, 14) .
As previously reported by others(9) , COS cells
transiently transfected with the wild type NTR expressed high levels of
-
I-BH-NT(2-13) binding sites, as determined on
membrane preparations (B
= 2240 ±
250 fmol/mg protein; Fig. 2). The affinity of
-
I-BH-NT(2-13) for the transfected receptors (K
= 0.45 nM) and the specificity
of the binding for a series of neurotensin analogs (data not shown)
were also consistent with previous reports(9, 14) .
When the binding experiments were carried out on whole cells, a large
fraction of the radioactivity specifically bound at 60 min remained
resistant to acid washes, indicating that it was internalized inside
the cells. Interestingly, no G-protein coupling of the receptor was
observed in these cells (data not shown), suggesting that receptor
sequestration/internalization still proceeds even if the receptors are
functionally uncoupled from the second messenger pathway. Similarly, in
the case of the
-adrenergic receptor(16) ,
functional coupling to G protein was shown not to be required for
agonist-induced internalization of the receptor.
Figure 2:
Binding affinity and internalization
efficiency of wild type and mutated neurotensin receptors expressed in
COS 7 cells. Data are expressed as the mean ± S.E. of at least
four separate experiments. The efficiency of internalization is
expressed as the ratio between internalized and total cell-associated
ligand at equilibrium, i.e. after incubation for 30 min at 37
°C. a, deletion of a cluster of Ser and Thr residues; b, deletion of a cluster of positively charged residues; c, point mutation. Binding experiments were carried out on
transfected cell membranes for 20 min at 25 °C. IC (nM) represents the concentration of the peptide that
inhibits 50% of the specific binding. The mean B
value obtained for both the wild type (Wt) and mutated
receptors is 2240 ± 250 fmol/mg protein. PheAsO,
phenylarsine oxide; BHNT, Bolton-Hunter
neurotensin.
The NTR possesses
several Ser and Thr residues within transmembrane domains 5 and 6.
Because earlier studies on the Hm1 muscarinic cholinergic receptor had
shown that Ser- and Thr-rich domains in the third cytoplasmic loop were
required for internalization(4) , we first tested the influence
of sequences that include these residues on the internalization of the
NTR. Deletions of three of these Ser- and Thr-containing sequences
(273-282,
287-296, and
299-305)
affected neither the affinity of mutated receptors for iodinated
neurotensin nor the efficiency of internalization of the
receptor-ligand complexes (Fig. 2).
Subsequently, our
investigation targeted the two potential palmitoylation sites Cys-386
and Cys-388. Indeed, homologous residues were previously found to be
involved in the internalization of the thyrotropin-releasing hormone
receptor(5) . Substitution of the Cys-386 and Cys-388 with
glycine residues (Fig. 1, CC386,388GG) in a full-length
mutant produced a protein able to bind and internalize
-
I-BH-NT(2-13) with properties identical to
those of the wild type NTR (Fig. 2). We conclude from these
results that if these two Cys residues (386, 388) can be palmitoylated
to form bridges with the plasma membrane, they are not implicated in
the internalization of the NTR.
In a third series of
experiments, we tested the involvement of the carboxyl-terminal tail in
the internalization of the NTR by truncating the receptor distal to the
Trp-391 residue (Fig. 1, R392stop). This mutated
receptor, in which a cluster of positive charges RRKKR and a series of
Thr and Ser residues were deleted, bound
-
I-BH-NT(2-13) with the same affinity
(IC
= 0.53 nM) as the unaltered NTR (Fig. 2). However, in cells transfected with this truncated
receptor, the level of ligand-induced internalization was markedly
reduced as compared with that observed in cells transfected with the
wild type (Fig. 2). This result indicates that, as previously
observed for several other GPCRs (5, 6, 7) ,
the structural information necessary for internalization of the NTR is
contained within the carboxyl terminus of the receptor.
In order to
more precisely localize the tail elements critical for the
internalization process, we made several deletions in the carboxyl tail
(394-398,
400-402,
407-411,
415-417,
418-423) of the NTR or truncated it
after the Glu-421 residue (Fig. 1, T422stop). All of
these receptors maintained the same affinity as the unaltered NTR for
-
I-BH-NT(2-13). However, three of these, the
415-417, the T422stop, and the
418-423 did show a
reduction of internalization efficiency; the greatest degree of
inhibition was observed in the case of the T422stop NTR ( Fig. 2and Fig. 3A). These results suggest that
only selective Ser and Thr in the carboxyl-terminal tail are involved
in the internalization of the NTR and that the last three
carboxyl-terminal residues (TLY) are especially critical.
Figure 3: Internalization kinetics of wild type and mutated neurotensin receptors expressed in COS 7 cells. Panel A, internalization rates for wild type (Wt) and truncated neurotensin receptors. Panel B, internalization rates for neurotensin receptors with point mutations on the carboxyl-terminal end. The amount of ligand internalization is expressed as the percent of total cell-associated ligand at each time. For both panels, each point represents the mean of at least three different experiments, with duplicate determinations. BHNT, Bolton-Hunter neurotensin.
To further identify which residues of the last three carboxyl termini are implicated in the internalization process, we substituted Thr-422 and Tyr-424 with a Gly, either separately or in combination. Substitution of Thr-422 did not affect the efficiency of neurotensin internalization, whereas substitution of Tyr-424 slightly decreased the amount of radioactive ligand internalized as compared with cells transfected with the native NTR (Fig. 3B). Maximal inhibition of the internalization was obtained with the mutant T422G,Y424G, which combined substitutions of Thr and Tyr residues ( Fig. 2and Fig. 3B). These results demonstrate that although neither Thr-422 nor Tyr-424 is individually critical for neurotensin-induced internalization, the integrity of these two residues should be preserved for maximal efficiency of the internalization process.
In order to determine if the addition of
the cytoplasmic internalization signal YTDI (17) to the
truncated T422stop NTR could restore the internalization properties of
this mutant, we incorporated the peptide sequence from the vesicular
stomatitis virus (VSV) containing the YTDI signal into the receptor
after the Glu-421 residue (Fig. 1). The corresponding
422-424-VSV mutant receptor bound iodinated NT with an
affinity identical to that of the wild type NTR but internalized as
poorly as its corresponding untagged receptor (Fig. 2). These
results indicate that the internalization of the NTR and the vesicular
stomatitis virus involves different sequences.
Confocal
microscopic analysis of the effect of a tail end NTR mutation on
internalization was carried out on COS 7 cells transfected with the
422-424-VSV tagged receptor using fluo-NT as a marker.
Fluo-NT is a fluorescein isothiocyanate-tagged derivative of
neurotensin, which was previously shown to bind with the same affinity
and selectivity as native neurotensin to the rat brain
NTR(15) . Examination of serial optical sections through cells
labeled with fluo-NT at 37 °C differed markedly depending upon the
type of receptor transfected. No labeling was observed on
nontransfected cells (results not shown). In cells transfected with the
wild type NTR, the labeling was mainly cytoplasmic and took the form of
small, rounded, and intensely fluorescent granules (Fig. 4a), whereas in cells transfected with the
modified NTR, the labeling remained confined to the cell surface where
it formed large ``hot spots'' (Fig. 4b). The
intracellular labeling in cells transfected with the wild type NTR
conformed to that observed in other cell types endowed with native NTRs (15) and likely reflects internalization of the ligand through
the endocytic pathway(18) . The fact that this pattern was no
longer apparent in cells transfected with the
422-424-VSV
mutant confirms that the
422-424-VSV tagged receptor lost
its capacity to be internalized. However, the preservation of
``hot spots'' on the membrane suggests that the receptor
retained its ability to aggregate on the cell surface after binding NT.
Figure 4:
Confocal microscopic imaging of COS 7
transfected with either the wild type (panel A) or the
422-424-VSV mutated form (panel B) of the rat
neurotensin receptor. Cells were incubated with 1 nM fluo-NT
for 30 min at 37 °C. Optical sections were taken at the cells
midheight and averaged over 32 scans/frame. Whereas the ligand is
clearly internalized in cells transfected with the wild type receptor,
it remains clustered on the cell surface in cells transfected with the
mutated form (arrows).
In conclusion, we have shown that the carboxyl-terminal cytoplasmic
tail of the NTR is essential for internalization. Although no unique
residue is absolutely necessary for this process to occur, residues
located at the very carboxyl-terminal end are the most important for
internalization. Thus, the order of increasing loss of sequestration
for our various deletion mutants is 394-398
399-405 =
400-402 <
407-411
<
415-417 <
418-423 = T422stop. The
most striking result is that substitution of both carboxyl-terminal
Tyr-424 and of Thr-422 with Gly is sufficient to prevent
internalization almost completely, although individual modification of
each one of these residues is without noticeable effect. Why both amino
acids should be needed for efficient internalization remains a matter
for speculation. One possibility is that these residues jointly
interact with a single intracellular component, for instance through
phosphorylation of their lateral side chains. Removal of a single amino
acid would be compensated for by the other, but deletion of the two
would abolish the interaction with the intracellular component and, by
way of consequence, the internalization. Comparison of our data with
those obtained for other G protein-linked receptors shows that no
general sequestration motif can be defined in terms of primary
structure. Since hydroxylated residues are often important for
internalization of this type of receptor, a special tridimensional
arrangement of Ser, Thr, and Tyr residues in the sequence could
constitute the structural element specifically recognized by the cell
for internalization.