(Received for publication, March 22, 1996, and in revised form, November 1, 1996)
From the Departments of Surgery,
Medicine and
** Physiology, University of California,
San Francisco, California 94143 and ¶ Khepri Pharmaceuticals
Inc., South San Francisco, California 94080
Although agonist-induced endocytosis of G-protein-coupled receptors is critical for receptor desensitization and resensitization, receptor motifs that interact with the endocytic apparatus have not been adequately characterized. We examined the effects of mutating the rat neurokinin-1 receptor on endocytosis using 125I-substance P, fluorescent substance P, and receptor antibodies. Substance P induced rapid internalization of wild-type receptors that were targeted to perinuclear endosomes. Truncation of the C-tail at residues 324, 342, and 354 reduced internalization up to 60% and caused retention of receptors at the cell surface and in superficial endosomes. Mutation of Tyr-341 and Tyr-349 in potential tyrosine-containing endocytic motifs of the C-tail also impaired internalization. A Y305A mutant within the putative NPX2-3Y endocytic motif of the seventh transmembrane domain showed impaired signaling and was minimally expressed at the plasma membrane but was found in cytoplasmic vesicles. In contrast, a Y305F mutant signaled normally and was normally expressed at the plasma membrane but showed impaired internalization. Thus, endocytosis of the neurokinin 1 receptor relies on several tyrosine-containing sequences in the C-tail and seventh transmembrane domain.
Receptor endocytosis is a fundamental process with many diverse functions. Endocytosis of some receptors, such as the transferrin receptor, is required for the uptake of nutrients by many cell types. These receptors internalize constitutively, without the need to bind ligand. In contrast, agonist binding is required for internalization of receptors for many hormones, neurotransmitters, and growth factors (1). Endocytosis of these receptors contributes to receptor desensitization, resensitization, and down-regulation, critical processes that regulate cellular responses.
The main pathway for constitutive and agonist-induced endocytosis is by
clathrin-coated pits, although alternative pathways involve non-coated
vesicles or caveolae (2). Clathrin-mediated endocytosis probably
requires interaction of specific receptor motifs with components of the
endocytic machinery (3). Tyrosine-containing motifs of six residues
forming a tight -turn are common, and interchangeable endocytic
signals are found in the intracellular domains of constitutively
internalized and tyrosine kinase receptors (1, 4). In constitutively
internalized receptors, they may be continuously exposed to the
endocytic machinery, whereas agonist-induced internalization of the
signaling receptors may require receptor modification to expose a
cryptic endocytic motif. Thus, a point mutation that inactivates the
tyrosine kinase activity of the epidermal growth factor receptor
abolishes agonist-induced internalization, probably by preventing
exposure of an endocytic motif (5). In contrast, deletion of the entire
tyrosine kinase domain abolishes the endocytic switch and results in
constitutive endocytosis, which is probably due to continuous exposure
of endocytic motifs.
Although regions in the C-tail and TMD1 VII
have been implicated as important for endocytosis of some
G-protein-coupled receptors (6-12), a common endocytic motif has not
been identified, and the nature of the molecular switch for
agonist-induced internalization is unknown. Agonist-induced
internalization may require activation of signaling mechanisms or
receptor phosphorylation. Arguing against the first possibility are
observations with receptor mutants that either signal normally and show
impaired internalization or exhibit impaired coupling to G-proteins and
normal internalization (13, 14). This suggests that different domains
are involved in internalization and G-protein coupling. Agonist binding
induces phosphorylation of Ser and Thr residues of many
G-protein-coupled receptors, and Ser/Thr-rich regions are critical for
internalization (6, 8-10, 15). However, phosphorylation of the
2-AR is not required for internalization (16-18).
Recently, there has been considerable interest in the mechanism and function of endocytosis of the NK1R, a G-protein-coupled receptor for the neuropeptide SP (19-22). SP is a neurotransmitter in the central and peripheral nervous systems, stimulates smooth muscle contraction and exocrine gland secretion, and induces neurogenic inflammation, actions that are mediated in part by the NK1R (23). SP stimulates clathrin-mediated endocytosis and recycling of the NK1R in multiple cell types (19-22). It is important to identify endocytic motifs of the NK1R since receptor endocytosis and recycling determine the ability of a cell to respond to SP (24). Although much is known about the extracellular and transmembrane domains of the NK1R that interact with receptor agonists and antagonists (25, 26), there is little information about NK1R domains that are necessary for endocytosis. Thus, the aim of this investigation was to identify motifs for SP-induced endocytosis of the NK1R. Based on our knowledge of endocytic motifs of single TMD receptors and on the limited information on G-protein-coupled receptors, we mutated the rat NK1R and investigated the effects of the mutations on agonist-induced endocytosis. We identified Tyr-containing motifs in the C-tail and TMD VII of the NK1R that are important for endocytosis and which resemble motifs of the single TMD receptors.
SP was labeled with Bis-functional cyanine 3.18 as described (27). A rabbit polyclonal antiserum (number 11884-5) was raised to a 15-residue peptide (K393TMTESSSFYSNMLA407), corresponding to the intracellular C terminus of the rat NK1R (28). A mouse monoclonal antibody (M2) to the Flag peptide (DYKDDDDK) was from International Biotechnologies, Inc. (New Haven, CT). A mouse monoclonal antibody (X22) to the heavy chain of clathrin was a gift from Dr. Francis Brodsky (University of California, San Francisco).
Mutagenesis of Rat NK1RMutants were generated by PCR using
the rat NK1R cDNA with an N-terminal Flag-peptide in pcDNAI/neo
as a template (28). The Flag epitope allowed detection of NK1R with the
Flag antibody as well as the antibody to the C terminus of the
receptor. The Flag does not alter the signaling or trafficking of the
NK1R (19, 28). Two overlapping fragments of the coding region were
amplified using SP1Flag
(5-GATCG
GCCACCATGGACCAAGG-3
,
HindIII-site underlined) as 5
primer and
NheI673B (5
-GGATCTC
CCACAGTG-3
) as 3
primer in one reaction, and NheI673A
(5
-CACTGTGG
GAGATCC-3
) and SP2
(5
-CATAA
CTAGGCCAGCATGTTAG-3
,
NotI-site underlined) in the other reaction. Primers
NheI673A and B introduced an NheI-site (underlined) at base 673 of the coding region by silent mutagenesis (mutated residues in bold). The reaction included 0.1 µg of template DNA, 0.1 µM of each primer, 50 µM each
dNTP, 10 × cloned Pfu buffer and 2.5 units Pfu polymerase in a
volume of 100 µl. The PCR conditions were 1 min at 94 °C, 2 min at
55 °C, and 3 min at 72 °C for 30 cycles. Both reactions were
combined and the products purified by ultrafiltration. The purified
fragments were used as templates in a PCR reaction with primers SP1Flag
and SP2. The product of this reaction was cut with
HindIII/NotI and cloned into pcDNA3. This
construct was used as NK1R-wt. All other mutations were introduced by
the aforementioned strategy using primers to the Sp6 and T7 regions in
pcDNA3 as flanking primers and other primers to introduce the
mutations. Truncated receptors were generated by amplifying rat NK1R
with SP1Flag and the primers
311,
324,
342, and
354 containing a stop codon and a NotI restriction site. The
fidelity of PCR amplification was verified by sequencing all constructs in both directions. All mutated receptors were cloned into
HindIII/NotI sites of pcDNA3.
We stably expressed receptors in KNRK cells, which do not express endogenous NK1R, using Lipofectin (28). Clones were selected in medium containing 800 µg/ml G418 and screened by 125I-SP and cy3-SP binding or NK1R immunofluorescence. Clones were maintained in medium containing 400 µg/ml G418. Cells were plated 24-48 h before experiments on poly-D-lysine-coated glass coverslips (for microscopy or measurement of Ca2+ mobilization) or on 35-mm plastic wells (for 125I-SP binding experiments, ~105 cells plated per well).
Binding and Endocytosis of 125I-SPCells were incubated in Hank's balanced salt solution, 0.1% bovine serum albumin, and 50 pM 125I-SP for 60 min at 4 °C, washed, and incubated at 37 °C for 0-10 min (19). An acid wash procedure was used to separate cell-surface (acid-sensitive) from internalized (acid-resistant) 125I-SP, exactly as described (19). The KD and Bmax were determined by competition binding and Scatchard analysis (28). Cells were incubated with 50 pM 125I-SP and 30 pM to 100 nM SP for 60 min at 4 °C. Nonspecific binding was measured by preincubating the cells with 1 or 2.5 µM SP and was subtracted to give specific binding.
Examination of Cy3-SP and NK1R Endocytosis by MicroscopyCells were incubated in Dulbecco's modified Eagle's medium, 0.1% bovine serum albumin, and 100 nM cy3-SP for 60 min at 4 °C, washed, and incubated at 37 °C for 0-30 min (19, 21). Cells were fixed with 4% paraformaldehyde in 100 mM phosphate-buffered saline for 20 min at 4 °C and mounted. Cy3-SP specifically binds to the NK1R in these cells (27). We also used an NK1R and Flag M2 antibodies to localize NK1R. Cells were incubated with 100 nM SP at 4 °C, washed, incubated at 37 °C for 0-30 min and fixed. NK1R and clathrin were localized by indirect immunofluorescence (19, 21). Cells were examined with an MRC 1000 Confocal Microscope using a Zeiss plan-Apochromat x100 oil-immersion objective. Images were collected at an aperture of 2-5 mm and a zoom of 2. Typically, 10-20 optical sections were taken at 0.5-µm intervals.
Measurement of Ca2+ MobilizationCells were washed with Hank's balanced salt solution, 0.1% bovine serum albumin and incubated with 2.5 µM Fura-2/AM for 20 min at 37 °C (28). Fluorescence was measured in a fluorimeter at 340- and 380-nm excitation and 510-nm emission. A single injection of SP (30 pM to 0.1 µM) was made. Results are expressed as the ratio of the fluorescence at 340 and 380 nm, which is proportional to [Ca2+]i.
Statistical AnalysisResults are expressed as mean ± standard error. Differences between multiple groups are examined by analysis of variance and Student-Newman Keuls test and between two groups by Student's t test. A p < 0.05 is considered statistically significant.
To identify regions of the NK1R that are important for
endocytosis, we generated a series of mutants (Fig.
1).
C-terminal Truncations
First, we progressively truncated the
C-tail (designating receptors as "" followed by number of the
last residue). The intracellular C-tail of the rat NK1R extends 99 residues from the plasma membrane (309-407). Tyr, Ser, and Thr
residues, usually 30-50 residues from the plasma membrane, have been
implicated in endocytosis of several G-protein-coupled receptors (8,
29-32). The C-tail of the NK1R contains three Tyr residues (Tyr-331,
Tyr-341, and Tyr-349) that are conserved between species and which may
be important for endocytosis. Tyr residues 341 and 349 are surrounded
by six Ser and Thr residues. We designed four truncated receptors to remove varying portions of the C-tail (Fig. 1):
354, lacking part of
the tail with no conserved Tyr residues;
342, lacking Tyr-349 and a
portion of the surrounding Ser and Thr residues;
324, lacking
Tyr-349, Tyr-341, Tyr-331 and the remaining Ser and Thr residues; and
311, in which the entire C-tail including the putative
palmitoylation site was deleted. NK1R-
311 corresponds to the
naturally truncated, potentially alternatively spliced NK1R variant
(33, 34). Immunofluorescence with the Flag antibody revealed that
NK1R-wt, NK1R-
354, NK1R-
342, and NK1R-
324 were normally
located at the plasma membrane (not shown). However, NK1R-
311 was
mostly found intracellularly in vesicles, suggesting mistargeting, and
was not further evaluated.
For all mutants, we initially measured the time course of receptor endocytosis using 125I-SP. Cells were incubated with 125I-SP for 60 min at 4 °C, washed, and incubated at 37 °C for 0, 5, or 10 min. They were washed with acid to separate cell surface from internalized label. We used these results to identify mutants with significant alterations in endocytosis. These were analyzed in more detail by measuring internalization of 125I-SP at 0, 1, 2, 4, or 8 min. The data points from 0, 1, and 2 min were fitted to a linear function; the initial rate of internalization was calculated from the slope and is expressed as % internalization/min.
When NK1R-wt cells were incubated with 125I-SP at 4 °C
and immediately washed with acid, 13.8 ± 1.7% of the
specifically bound counts were in the acid-resistant, internalized
fraction. After 5 min at 37 °C, 57.2 ± 2.0% of specific
counts were internalized. The initial internalization rate was 9.9 ± 0.9%/min (r2 for linear regression = 0.995) (Fig. 2A). At 4 °C, 8-20% of
specific counts were internalized by NK1R-354, -
342, or -
324
cells. After 5 min the proportion of specific counts that were
internalized was 50.6 ± 0.5% for NK1R-
354, 21.1 ± 0.8%
for NK1R-
342, and 32.8 ± 3.5% for NK1R-
324, significantly
less than for NK1R-wt (p < 0.05 compared with NK1R-wt,
Table I). These differences were also significant at 10 min. The initial rates of internalization were measured for NK1R-
342
and NK1R-
324 cells, since they showed the largest defects in
endocytosis. The initial rat of internalization was 2.4 ± 0.5%/min (r2 = 0.999) for NK1R-
342 cells and
3.0 ± 0.6%/min (r2 = 0.971) for
NK1R-
324 cells, significantly slower than for NK1R-wt cells
(p < 0.05 compared with NK1R-wt, Fig.
2A).
|
We used cy3-SP, immunofluorescence, and confocal microscopy to
determine the effects of the mutations on the subcellular distribution of NK1R. We have shown that cy3-SP and NK1R are co-localized in the
same endosomes up to 30 min after internalization in KNRK cells
expressing NK1R-wt (19-21). Thus, cy3-SP can be used to localize the
NK1R at these times. When NK1R-wt cells were incubated with cy3-SP at
4 °C and immediately fixed, cy3-SP was confined to the plasma
membrane (Fig. 3). After 2 min at 37 °C, there was
minimal detectable surface labeling, and cy3-SP was found in
superficial endosomes. After 5 min, cy3-SP was mostly present in
perinuclear endosomes (Fig. 3), and the distribution was similar at 10 and 30 min. In NK1R-354, -
342, and -
324 cells at 4 °C,
cy3-SP was confined to the plasma membrane (Fig. 3). In NK1R-
354
cells, a substantial amount of cy3-SP remained at the plasma membrane after warming, and endosomes containing cy3-SP remained in a
superficial location and did not proceed to a perinuclear region (Fig.
3). The largest defect in internalization was observed in NK1R-
342 cells, in which cy3-SP was retained at the plasma membrane and was
rarely detected in endosomes even after 30 min (Fig. 3). In NK1R-
324
cells, cy3-SP was also detected at the plasma membrane at all time
points and there was minimal internalization (Fig. 3).
We assessed the function of the NK1R-wt and the mutated receptors by
measuring SP-induced changes in [Ca2+]i and
determining the EC50 and maximal response. In NK1R-wt
cells, SP induced a prompt increase in [Ca2+]i
with an EC50 of 0.55 nM (Table I). SP also
increased [Ca2+]i in NK1R-354, -
342, and
-
324 cells, but SP was more potent, and the dose-response curves
were shifted to the left between 3- and almost 7-fold (Fig.
4A, Table I). Maximal responses were similar
for NK1R-wt and the truncated receptors. Those receptors with the
largest defects in internalization were further characterized by
determining the KD and Bmax
of the receptors. The KD for 125I-SP
binding was 7.4 ± 1.6 nM and the
Bmax was 58.4 ± 4.9 fmol/105
cells for NK1R-wt (Table I). The KD and
Bmax were similar in NK1R-
342 and -
324
cells. Thus, although we do not know the reason for increased
sensitivity of truncated receptors to SP, their impaired ability to
internalize cannot be explained by defective signaling, and defects in
ligand binding cannot explain the impaired endocytosis of these
truncated receptors.
Tyrosine Mutations in the C-tail
Tyr-containing endocytic motifs, in which the Tyr is of critical importance, have been identified for many of the single TMD proteins (1, 32). We investigated potential Tyr-containing internalization motifs in the C-tail of the NK1R by mutating conserved residues. Individual mutation of the conserved tyrosines (Tyr-331, Tyr-341, and Tyr-349) in the C-tail to Ala significantly reduced the extent of internalization of 125I-SP but to different degrees. Thus, after 5 min at 37 °C, 50.5 ± 2.3% of specific counts were internalized by NK1R-Y331A, 48.1 ± 3.8% by NK1R-Y341A cells, and 40.6 ± 3.9% by NK1R-Y349A cells, compared with 57.2 ± 2.0% for NK1R-wt cells (p < 0.05 compared with NK1R-wt, Table I). The greatest effect, for the Y349A mutant, represents a reduction in the extent of internalization of 30%. A double Tyr-341 and Tyr-349 mutation did not have an additive effect on attenuation of endocytosis. The initial internalization rates were 6.3 ± 0.8%/min (r2 = 0.980) for NK1R-Y341A and 4.0 ± 0.6%/min (r2 = 0.981) for NK1R-Y349A cells and, therefore, significantly slower than for NK1R-wt cells (9.9 ± 0.9%/min) (p < 0.05 compared with NK1R-wt, Fig. 2B).
At 4 °C, cy3-SP was confined to the plasma membrane in all mutants
involving Tyr residues in the C-tail (NK1R-Y341A and NK1R-Y349A, Fig.
5; NK1R-Y331A and NK1R-Y341A/Y349A not shown). After 5 min at 37 °C, cy3-SP was retained at the plasma membrane and found in superficial and perinuclear endosomes in cells expressing NK1R-Y331A (not shown) and NK1R-Y341A (Fig. 5). In NK1R-Y349A cells, cy3-SP was
mostly retained at the plasma membrane and in superficial endosomes
after 5 min, although there were a few perinuclear vesicles containing
cy3-SP (Fig. 5). This is in contrast to NK1R-wt cells, in which cy3-SP
was mostly found in many perinuclear endosomes after 5 min (Fig.
3).
EC50 values for SP-induced Ca2+ mobilization ranged from 0.037 to 0.156 nM for NK1R-Y331A, -Y341A, and -Y349A cells compared with 0.55 nM for the NK1R-wt (Fig. 4B, Table I). Maximal Ca2+ responses were similar. The KD and Bmax were similar to NK1R-wt except for the Y341 mutant, where the KD was ~4-fold lower. Thus, defects in endocytosis are unlikely to be the result of markedly abnormal binding and signaling.
Tyrosine Mutations in TMD VIIA Tyr-containing motif (NPX2-3Y) in TMD VII is highly conserved within G-protein-coupled receptors and may be a common endocytic motif (7). We examined this possibility for the NK1R by mutating Tyr-305 in TMD VII to Phe or Ala. Phe can replace Tyr in many, but not all, proteins without a significant effect on internalization, whereas Ala substitutions usually abolish the signal (1).
Mutation of Tyr-305 to Phe significantly reduced the extent of endocytosis of NK1R, as measured by internalization of 125I-SP. Thus, after 5 min at 37 °C, only 40.3 ± 4.7% of specific counts were internalized by NK1R-Y305F cells, which represents a reduction of internalization by 30% compared with NK1R-wt cells (p < 0.05 compared with NK1R-wt, Table I). Mutation of Tyr-305 to Ala resulted in increased internalization at 4 °C, although there was low specific binding compared with the other mutants. After 60 min at 4 °C, 29.4 ± 7.1% of specific counts were internalized in NK1R-Y305A cells, compared with 13.8 ± 1.7% in NK1R-wt cells (Table I). However, after 5 min at 37 °C, the proportion of internalized 125I-SP was similar for NK1R-Y305A and NK1R-wt cells. The initial internalization rates were 7.9 ± 1.1%/min (r2 = 1.000) for NK1R-Y305F cells and 7.1 ± 1.0%/min (r2 = 0.921) for NK1R-Y305A cells (Fig. 2C). These rates were slower than for NK1R-wt cells but not statistically significant different.
At 4 °C, cy3-SP was confined to the plasma membrane of NK1R-Y305F
cells (Fig. 6). In contrast, there was only very weak
surface binding of cy3-SP in NK1R-Y305A cells, and cy3-SP was also
detected in vesicles at 4 °C (Fig. 6). In NK1R-Y305F cells after 5 min at 37 °C, cy3-SP remained at the cell surface and in superficial endosomes, indicating diminished internalization of NK1R-Y305F compared
with NK1R-wt (Fig. 6). However, in NK1R-Y305A cells, cy3-SP was
found in perinuclear endosomes after 5 min (Fig. 6).
The low surface binding and apparent internalization of
125I-SP and cy3-SP by NK1R-Y305A cells at 4 °C prompted
us to directly localize the NK1R by immunofluorescence with an antibody
to the C-tail of the receptor. When NK1R-wt and NK1R-Y305F cells were incubated with SP at 4 °C and immediately fixed, receptor
immunoreactivity was mostly confined to the plasma membrane with very
few immunoreactive endosomes (Fig. 7). In sharp
contrast, in NK1R-Y305A cells at 4 °C, receptor immunoreactivity was
mostly found in perinuclear vesicles with minimal surface labeling
(Fig. 7). After 10 min at 37 °C, receptor immunoreactivity was
detected in larger perinuclear vesicles in NK1R-wt cells, whereas
immunoreactivity was also retained at the plasma membrane in NK1R-Y305F
cells (Fig. 7). The subcellular location of receptor immunoreactivity
was similar in NK1R-Y305A cells at 4 °C and at 37 °C (Fig.
7).
In NK1R-Y305F cells, the EC50 and efficacy of SP-induced
Ca2+ mobilization were similar to NK1R-wt cells (Fig.
4C, Table I). However, SP was over 4-fold less potent and
7-fold less efficacious in NK1R-Y305A than in NK1R-wt cells (Fig.
8, Table I). The KD values for
NK1R-Y305 and NK1R-Y305A were similar to NK1R-wt, but the
Bmax for NK1R-Y305A cells was markedly lower.
Thus, the Y305F mutation does not affect SP-induced Ca2+
mobilization, whereas the Y305A mutation interferes with
Ca2+ mobilization.
Serine/Threonine-Mutations and a Mutation of the EMKST Motif in the C-tail
Ser/Thr-rich regions are critical for internalization of
several G-protein-coupled receptors (6, 8-10, 15). Based on our
results with the truncated receptors and on studies suggesting that Ser
and Thr residues in the vicinity of Tyr residues may be a more common
determinant of internalization signals than their possible location in
a consensus site for phosphorylation (6, 12), we mutated Ser and Thr
residues in the C-tail of the NK1R. We also mutated Lys-337 because it
is situated in a EMKST motif, which closely resembles the
internalization signal DAKSS of the yeast -pheromone receptor (35).
Mutation of the lysine in this motif prevents endocytosis.
The time course of internalization of 125I-SP by cells
expressing NK1R-S338G/T339A, NK1R-T339A/S347A, and NK1R-K337A was the same as for NK1R-wt cells (Table I). Mutation of all Ser and Thr
residues between 338 and 352 (NK1R-ST338-352) slightly enhanced internalization after 5 min at 37 °C but not after 10 min (Table I).
The initial internalization rate for NK1R-
ST338-352 cells was
13.9 ± 1.4%/min (r2 = 0.998), higher than
in NK1R-wt cells, but the difference did not reach statistical
significance (Fig. 2D).
Cy3-SP was confined to the plasma membrane at 4 °C for all mutants
(Fig. 9). Surprisingly, after 5 min at 37 °C cells
expressing NK1R-ST338-352 demonstrated a halo-like retention of
cy3-SP in small peripheral vesicles, seemingly in contrast to the data
obtained by using 125I-SP. To a lesser extent, this
phenomenon was also detectable in cells expressing NK1R-S338G/T339A.
Compared with NK1R-wt, the amount of cy3-SP in perinuclear endosomes
was not markedly reduced in these mutants. Endocytosis of cy3-SP by
cells expressing NK1R-T339A/S347A (Fig. 9) and NK1R-K337A (not shown)
was similar to that observed in NK1R-wt cells. Cells expressing
NK1R-S338G/T339A, NK1R-
ST338-352, and NK1R-K337A responded to SP
with EC50 values for Ca2+ mobilization that
were similar to NK1R-wt cells (Fig. 4D, Table I). In cells
expressing NK1R-T339A/S347A, SP was about 3-fold less potent for
Ca2+ mobilization. The KD for
NK1R-
ST338-352 was similar to NK1R-wt. Despite the reduced
Bmax of this mutant, the efficacy for
Ca2+ mobilization was similar to NK1R-wt.
Effects of Mutations on the Mechanism of Endocytosis
To
determine if the mutated receptors with abnormal rates of endocytosis
were internalized similarly to NK1R-wt at sites of clathrin (21), we
colocalized cy3-SP and clathrin. Cells were incubated with cy3-SP for
60 min at 4 °C, washed, incubated at 37 °C for 2 or 5 min, and
fixed, and clathrin was localized by immunofluorescence. In cells
expressing NK1R-wt, most superficial vesicles containing cy3-SP were
initially stained with the clathrin antibody, as indicated by
superimposition of confocal images, where yellow denotes colocalization
(Fig. 10). In contrast, cy3-SP was mostly retained at
the cell surface of cells expressing truncated receptors such as
NK1R-342, and there was no clathrin colocalization (Fig. 10).
Although cy3-SP was internalized more slowly in cells expressing
NK1R-Y349A, NK1R-Y341A, or NK1R-Y305F, superficial vesicles containing
cy3-SP were still stained with the clathrin antibody (Fig. 10). In
cells expressing NK1R-Y305A, there were few superficial vesicles
detected containing cy3-SP, although these were occasionally stained
with the clathrin antibody (Fig. 10). In cells expressing
NK1R-
ST338-352, cy3-SP was retained in numerous small, very
superficial vesicles that were also stained by the clathrin antibody
(Fig. 10). Thus, despite the finding that many mutants affected the
internalization rate, endocytosis still proceeded at sites of
clathrin.
The combination of receptor mutagenesis, analyses using radiolabeled and fluorescently labeled SP, and use of antibodies to the NK1R and clathrin permitted detailed investigation of endocytic motifs of the NK1R. The results show that there are several sequences within the C-tail and TMD VII of the rat NK1R that are important for SP-induced endocytosis. We compared SP-induced endocytosis of mutant and wt NK1R stably expressed in KNRK cells. We have previously examined trafficking of the NK1R in KNRK cells in detail and found that it behaves like the NK1R in neurons and endothelial cells (19-21, 36).
The C-Tail of the NK1R Contains Endocytic MotifsTruncation
of the NK1R at residue 355 had a relatively small effect on
internalization, indicating that either residues 355 to 407 are not
critical for endocytosis or this region contains positive and negative
endocytic signals whose influences are offset when both are removed.
Further deletion at residue 342 markedly slowed the rate of endocytosis
and trafficking to a perinuclear region, indicating that more proximal
regions of the C-tail are critical for internalization. The
dramatically reduced rate of internalization of NK1R-342 compared
with NK1R-
354 or NK1R-wt indicates that residues 343-354 include an
important endocytic domain. Thus, the NK1R resembles the receptors for
gastrin-releasing peptide, thyrotropin-stimulating hormone, angiotensin
II, and parathyroid hormone, in which truncation of the C-tail also
decreases the rate of internalization (8, 12, 37, 38). In contrast, removal of the C-tail of the luteinizing hormone/chorionic gonadotropin receptor and the avian
1- or
2-AR
increases the rate of internalization (29, 39, 40). Further deletion of
residues 325 to 342 (NK1R-
324) slightly enhanced the degree of
endocytosis compared with NK1R-
342. This may be explained by the
presence of a negative endocytic signal between residues 325 and 342. Such a signal (EVQ) is found at the membrane-cytoplasmic interface of
TMD VII of the parathyroid hormone receptor (12). Alternatively,
incremental shortening of the C-terminal tail may increase the lateral
mobility of the receptor in the plasma membrane and, therefore, its
probability of becoming trapped in a clathrin-coated pit (41). With the exception of NK1R-
311, all of the truncated receptors were
appropriately expressed at the plasma membrane. This lack of surface
expression may explain the poor functional responses of the naturally
occurring truncated NK1R to SP (33, 42). Similar mistargeting occurs for other receptors truncated N-terminal to the putative palmitoylation site or lacking the entire C-tail (12, 29).
Tyr-containing endocytic motifs, in which the Tyr is of
critical importance, have been identified for many of the single TMD proteins (1, 32). They usually contain six residues forming an exposed
-turn. Positions 1, 3, and 6 are frequently occupied by aromatic or
large hydrophobic residues, with positions 3 and 6 more important to
the function of the motif than position 1. Either position 3 or 6 must
contain an aromatic residue, and residues in positions 2, 4, and 5 tend
to be polar and are often found in turns.
All of the conserved Tyr residues in the C-tail of the NK1R contribute
to endocytosis, since each mutant showed defective internalization,
albeit to different degrees. Mutation of Tyr-349 had the largest effect
on internalization. This is located between residues 343 and 354, and
removal of these residues in NK1R-342 resulted in the greatest
defect in internalization between the three truncated mutants. Tyr-341
and Tyr-331 are probably part of signals of lesser importance. However,
only the residues surrounding Tyr-331 fit the typical consensus
sequence for Tyr endocytic motifs, with aromatic or large hydrophobic
residues in positions 3 and 6 (GD331YEGL). Tyr-349 could be
placed in a hexapeptide motif with Tyr-349 in position 1 and Val and
Leu in positions 3 and 6 (349YKVSRL). Tyr-341 is not
surrounded by any other aromatic or large hydrophobic residues that fit
a 1-3-6 pattern. Tyr-containing motifs in the C-tail of other
G-protein-coupled receptors, such as the parathyroid hormone receptor
and angiotensin II1a receptor, are also important for
internalization (9, 12). However, mutation of surrounding residues and
the exchange of motifs between G-protein-coupled and single TMD
receptors will be required to determine the extent to which these
motifs resemble the Tyr-containing endocytic motifs of single TMD
receptors. In contrast, Tyr residues in the C-tails of the
2-AR and m2 muscarinic receptor are not critical for internalization (43, 44).
The NK1R-ST338-352 was internalized marginally more quickly than
NK1R-wt in response to SP. Thus, in contrast to some other G-protein-coupled receptors, in which Ser/Thr-rich regions are critical
for internalization (6, 8-10, 15), the ST338-352 region plays only a
subtle role in internalization of the NK1R.
A Tyr-containing motif (NPX2-3Y) in TMD VII is highly conserved within the G-protein-coupled receptors. It closely resembles endocytic motifs of the low density lipoprotein and insulin receptors, both of which fit the six-residue consensus sequence for Tyr-containing endocytic motifs (1, 32). It has been suggested that this may be a common endocytic motif for G-protein-coupled receptors (7).
We investigated this possibility by mutating this region of the NK1R. The NK1R-Y305F and NK1R-Y305A mutants behaved very differently. The conservative Y305F mutation did not affect the subcellular distribution of the receptor in the basal state after binding SP at 4 °C but caused diminished internalization after incubation at 37 °C. The more drastic Y305A mutation markedly altered the subcellular distribution of the receptor so that it was in intracellular vesicles even in the basal state. This could be due to mistargeting of newly synthesized receptors to the plasma membrane. However, several observations indicate that some receptors reach the plasma membrane. First, there was a low level of detectable binding of 125I-SP and cy3-SP to the cell surface at 4 °C. Second, there was internalization of 125I-SP after warming at a similar rate as for the NK1R-wt. Finally, the prominent intracellular pools of cy3-SP observed after incubation at 37 °C must have undergone endocytosis, since SP is hydrophilic and can only bind surface receptors. An alternative explanation for the detection of NK1R-Y305A in intracellular vesicles in the basal state is that this receptor internalizes constitutively. In support of this possibility is the elevated internalization of 125I-SP and cy3-SP at 4 °C. Thus, the Y305A mutation may induce a conformational change in the receptor that permits its interaction with the endocytic apparatus at 4 °C or even without agonist binding. If this is the case, the NPX2-3Y sequence in TMD VII could resemble the switch function of the tyrosine kinase domain for agonist-induced internalization of the epidermal growth factor receptor (5). The tyrosine kinase point mutation and the Y305F mutation may interfere with agonist-induced changes in receptor conformation, thereby preventing exposure of cryptic endocytic motifs. In contrast, deletion of the tyrosine kinase domain and the Y305A mutation may cause the receptors to assume a conformation in which endocytic motifs are constitutively exposed. Further experimentation is required to test this hypothesis.
Mutation of the TMD VII NPX2-3Y domain also affected signaling. Although the Y305F mutation did not affect SP-induced Ca2+ mobilization, both potency and efficacy of SP were reduced in NK1R-Y305A cells as compared with NK1R-wt cells. The impaired signaling of NK1R-Y305A may be explained by its low level of expression at the cell surface. Alternatively, the NPX2-3Y motif may have induced a conformational change in the receptor that alters its ability to interact with mechanisms of signal transduction.
The role of the NPX2-3Y domain in endocytosis
and signaling has been examined for other receptors. Mutation of the
corresponding Tyr in TMD VII of the 2-AR to Ala (Y326A)
abolishes internalization, reduces agonist-induced phosphorylation by
G-protein receptor kinase 2, and depresses activation of adenylyl
cyclase (7, 45, 46). Overexpression of this kinase rescues receptor
phosphorylation and internalization. Thus, the
2-AR-Y326A mutation alters the ability of the
agonist-occupied receptor to achieve a conformation required for
phosphorylation, signaling, and internalization. The more conservative
Y326F mutation of the
2-AR reduces internalization by
only 25% and also depresses phosphorylation and activation of adenylyl
cyclase (45). A tyrosine to alanine mutation of the
NPX2-3Y sequence in TMD VII of the angiotensin
II1A receptor also reduces internalization by ~25% (9, 11).
In contrast, an equivalent mutation does not affect endocytosis of the
receptor for gastrin releasing peptide (47).
These observations and the results of the present study suggest that the NPX2-3Y sequence of TMD VII is not an endocytic motif but may be important for maintaining the appropriate conformation of the receptor for endocytosis or signaling to occur. Therefore, Tyr-305 of the NK1R may be critical for transducing agonist binding at the outer face of the receptor into a conformational change in the C-tail that is necessary to trigger endocytosis or signaling. Recent structural models of G-protein-coupled receptors suggest that the NPX2-3Y motif is in an appropriate location to receive a signal from agonist-induced conformational changes in the ligand-binding region (48).
Mechanisms of SP-Induced Endocytosis of the NK1RThe NK1R internalizes by a clathrin-mediated mechanism (21), although some G-protein-coupled receptors internalize by caveolin-dependent pathways (49, 50) or by mechanisms that are independent of clathrin and caveolin (51). Despite the finding that many of the mutations affected the rate of internalization, or even resulted in retention of the receptor in very small, superficial vesicles, many of these internalized vesicles were colocalized with clathrin at early time points. Thus, the effect of the mutations is to alter the rate of clathrin-mediated endocytosis, rather than to divert the receptor into a different endocytic pathway. Further analyses will be required to identify proteins that interact with potential endocytic domains of the NK1R. However, Tyr-containing motifs in the C-tails of some membrane proteins interact directly with the clathrin-associated protein complex AP-2 (52, 53). The µ2-chain of AP-2 interacts with the SDYQRL motif of the C-tail of the integral membrane protein TGN38, as well as with similar motifs from lamp-1, CD68, H2-Mb, and the transferrin-receptor (54). The importance of the interaction between Tyr-containing endocytic motifs and the AP-2 complex has recently been questioned, since deletion of the high affinity AP-2 binding site in the C-tail of the epidermal growth factor receptor abolishes AP-2 binding to the receptor without affecting internalization (55).
ConclusionsMultiple domains in the intracellular C-tail and TMD VII of the NK1R are important for SP-induced endocytosis. All of the conserved Tyr residues in the C-tail participate in endocytosis although Tyr in positions 341 and 349 are most important. Although mutation of the Tyr residue of the NPX2-3Y sequence in TMD VII alters endocytosis, it is probable that this region is important for maintaining the correct conformation of the receptor and that it is not an endocytic motif. None of the point mutations reproduced the marked inhibition of endocytosis observed with the truncated receptors or abolished internalization, emphasizing that NK1R internalization relies on multiple endocytic motifs.
We thank Michelle Lovett and Patrick Gamp for expert technical assistance.