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INTRODUCTION |
Agonist-induced trafficking of G-protein-coupled receptors
(GPCRs)1 and associated
proteins plays an essential role in signal transduction (reviewed in
Refs. 1 and 2). Within seconds of stimulation, G-protein receptor
kinases and second messenger kinases translocate from the
cytoplasm to the plasma membrane, where they phosphorylate receptors
(3-5).
-Arrestins are multifunctional proteins that interact with
G-protein receptor kinase-phosphorylated receptors at the plasma
membrane. This interaction disrupts association of GPCRs with
heterotrimeric G-proteins and thereby mediates uncoupling and
desensitization (6-8).
-Arrestins also serve as adaptors for
clathrin-mediated endocytosis of certain GPCRs, which contributes to
desensitization by depleting the cell surface of receptors (9-13). In
addition,
-arrestins are scaffolds that recruit components of the
MAP kinase cascade into endosomes, which is necessary for mitogenic
signaling (14-16). Once internalized, GPCRs recycle to the cell
surface, which mediates resensitization (17-20), or are degraded in
lysosomes, which down-regulates receptors (21-24). However, despite
the importance of receptor trafficking for signal transduction, the
molecular mechanisms are not fully understood. Most information derives
from studies of very few receptors and is not necessarily applicable to
all GPCRs. Furthermore, most observations are made using transfected
cells expressing very high levels of GPCRs, which need not represent
cells that naturally express these receptors at physiological levels.
We investigated the mechanisms and function of substance P (SP)-induced
endocytosis of the neurokinin-1 receptor (NK1R) in transfected cell
lines and enteric neurons that naturally express this receptor. An
understanding of the mechanisms that regulate this system is important,
because SP and the NK1R mediate pain, depression, inflammation, smooth
muscle contraction, and exocrine secretion (25, 26). SP induces
endocytosis and recycling of the NK1R in neurons and endothelial cells
that participate in pain and inflammation (11, 18, 27-31). However,
the molecular mechanisms of this trafficking and its importance in
signal transduction are unknown.
We examined the role of the GTPases dynamin and ras-related GTPase 5a
(Rab5a) in SP-induced endocytosis of the NK1R and determined the
importance of this trafficking for desensitization, resensitization, and mitogenic signaling. The cytosolic GTPase dynamin mediates the
first steps of endosome formation at sites of clathrin-coated pits and
caveoli (32, 33). Dynamin is required for constitutive endocytosis of
the transferrin receptor as well as agonist-induced endocytosis of some
GPCRs (12, 13, 34-38). However, some GPCRs internalize by
dynamin-independent processes (12, 37, 39, 40). Rab5a mediates later
steps of formation of endosomes containing the transferrin receptor and
is required for endosomal translocation to a perinuclear region
(41-43). However, very little is known about the role of Rab5a in
trafficking of GPCRs other than recent observations that Rab5a
participates in the formation of endosomes containing the dopamine D2
and
2-adrenergic (
2-AR) receptors (38,
44) and contributes to down-regulation of the
-opioid receptor (24).
Nothing is known about the role of dynamin and Rab5a in trafficking of
the NK1R, and the relative roles of dynamin and Rab5a in
desensitization, resensitization and mitogenic signaling of GPCRs are unknown.
Our aims were to (a) determine the role of dynamin and Rab5a
in SP-induced endocytosis and intracellular trafficking of the NK1R by
expressing dominant negative mutants of these GTPases; (b)
compare the importance of dynamin and Rab5a-dependent
trafficking for desensitization and resensitization of signal
transduction; (c) define the role of this trafficking for
mitogenic signaling; and (d) establish whether dynamin and
Rab5a could contribute to trafficking of the NK1R in neurons that
naturally express this receptor.
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EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Enhanced green fluorescent protein
(EGFP) expression vector pEGFP-C1, expression vector pIRES2-EGFP, and
JM109 bacteria were from CLONTECH (Palo Alto, CA).
Restriction enzymes, T4 ligase, and Lipofectin were from Life
Technologies, Inc. or New England Biolabs, Inc. (Beverly, MA). Exp Tag
DNA polymerase was from Stratagene (La Jolla, CA). A QiaEx extraction
kit was from Qiagen (Hilden, Germany). G418 was from Gemini
Bio-Products, Inc. (Calabashes, CA). Kanamycin and protease inhibitor
mixture were from Calbiochem. Glutathione-agarose was from Amersham
Pharmacia Biotech. The ECL detection kit was from Amersham Pharmacia
Biotech. The Alexa594 protein labeling kit was from Molecular Probes,
Inc. (Eugene, OR). SP or the NK1R-selective agonist
[Sar9,MetO211]SP was labeled with
Alexa594 as described (45). 125I-SP (2,000 µCi/mol) was
from Amersham Pharmacia Biotech. Antibodies to Rab5a, dynamin,
extracellular signal-regulated kinases 1 and 2 (ERK1/2), and
phosphorylated ERK1/2 (pERK1/2) were from Transduction Laboratories
(Santa Cruz, CA). The sources of other reagents and of antibodies to
FLAG and 12CA5 epitopes, NK1R, EGFP, and fluorescence- or
enzyme-tagged secondary antibodies have been described (11, 22, 23,
46-48).
Generation of Rab5a-GFP and HA-Dynamin Constructs--
Canine
Rab5a and the GTPase-defective binding mutant Rab5aS34N were fused to
EGFP at the N terminus by PCR using the forward primer
5'-GGCCGGAATTCCATGGCTAATCGAGGAGCAACA-3' (EcoRI
site underlined followed by translation initiation site) and the
reverse primer 5'-GGCCGGGGATCCTTAGTTACTACAACACTGACTCCT-3'
(BamHI restriction site underlined followed by stop codon).
Rab5a cDNA was used as template to amplify an 800-bp fragment that
was separated on an agarose gel and purified using a QiaEx extraction
kit. The PCR fragment and the vector pEGFP-C1 were digested with
EcoRI and BamHI and ligated with the T4 ligase
overnight at 16 °C. Chemically competent JM109 bacteria were
transformed using the heat shock method and selected on LB medium
containing 30 µg/ml of Kanamycin. Correct sequences were verified.
Rab5a tagged with EGFP is fully functional (42). Generation and use of
pcDNA3.1-HA-dynamin I and HA-dynamin I K44E (N-terminal HA.11
epitopes), a dominant negative mutant that lacks GTPase activity, have
been described (34). HA-dynamin-pIRES-EGFP constructs were generated by
enzymatic digestion of pcDNA3.1-HA-dynamin with XhoI and
XbaI and insertion of DNA into pIRES2-EGFP. The pIRES-EGFP
construct enabled convenient identification of transfected cells using
EGFP while avoiding attachment of EGFP to dynamin, which impairs its
function (49).
Generation of Transfected Cells--
Kirsten murine sarcoma
virus-transformed rat kidney epithelial cells (KNRK) were from the
American Type Tissue Culture Collection (Manassas, VA). Generation of
KNRK cells stably expressing rat NK1R with N-terminal FLAG epitope
(KNRK-FLAGNK1R cells), which does not affect receptor function, has
been described (50). To generate a cell line stably expressing FLAGNK1R
plus Rab5a-GFP or Rab5aS34N-GFP, KNRK-FLAGNK1R cells were transfected
with cDNA encoding Rab5a-GFP or Rab5aS34N-GFP (11).
Clones were screened by fluorescence microscopy and flow cytometry to
detect NK1R and Rab5a. Cells were also transiently transfected with
Rab5a and dynamin, since stable expression of dominant negative dynamin caused cell death, and to enable simultaneous comparison of transfected and untransfected cells by microscopy. KNRK-FLAGNK1R cells were transiently transfected by overnight incubation with 5 µg/ml cDNA encoding Rab5a-GFP, Rab5aS34N-GFP, HA-dynamin, or HA-dynamin K44E by
lipofection. The medium was replaced, and cells were studied 24 h
later. To transiently express HA-dynamin-pIRES-EGFP or HA-dynamin K44E-pIRES-EGFP, KNRK-FLAGNK1R cells were transfected by
electroporation (Bio-Rad) and plated for 18 h, and positive
transfected cells were sorted and plated 24 h before experiments.
Cell lines are designated KNRK-NK1R+Rab5a, KNRK-NK1R+Rab5aS34N,
KNRK-NK1R+DYN, or KNRK-NK1R+DYNK44E (where DYN represents dynamin).
Cells were prepared for experiments as described (11, 27) and were
usually incubated with 5 mM sodium butyrate for 18 h
before use to boost expression of transfected genes. As a control,
KNRK-FLAGNK1R cells were transfected with vectors without the Rab5a or
dynamin inserts. Expression of empty vectors or EGFP alone did not
affect signaling or trafficking of the NK1R (see Ref. 11; data not
shown) or related receptors (23).
Flow Cytometry--
Flow cytometry was used to monitor
expression of NK1R, Rab5a, and dynamin and to enrich populations of
transfected cells (11, 23). To detect the NK1R, cells were resuspended
in 1 ml of Iscove's medium containing 1 mg/ml bovine serum albumin,
with 10 µg/ml M2 antibody to the extracellular FLAG for 1 h at
4 °C, washed, and incubated with 2 µg/ml phycoerythrin-conjugated
goat anti-mouse IgG for 1 h at 4 °C. Expression of other
constructs was determined using EGFP. Cells were analyzed using a
Facscan flow cytometer (Becton Dickinson Co., Franklin Lakes, NJ).
Fluorophores were excited at 488 nm, and emission was collected at
530/30 nm for EGFP and 575/25 nm for phycoerythrin.
Western Blotting--
Western blotting was used to confirm
expression of Rab5a and dynamin (11, 23). Cells (107) were
pelleted and lysed in 1 ml of Laemmli buffer. Lysates were fractionated
by SDS-PAGE (4-15%), and proteins were transferred to nitrocellulose.
Membranes were incubated with 5% nonfat milk in 100 mM
PBS, pH 7.4, overnight and with antibodies to EGFP (9708; 1:1,000-1:20,000, 8 h, 4 °C), Rab5a, or dynamin (1:1,000,
2 h, room temperature). Membranes were washed and incubated with
goat anti-rabbit IgG conjugated to horseradish peroxidase (1:8,000, 1 h, room temperature). Proteins were detected on film using an ECL detection kit. Controls included preabsorption of the diluted primary antibody with the EGFP fusion protein (1-2 µg/ml) for 1 h at 37 °C and use of nontransfected KNRK cells.
Endocytosis of 125I-SP--
The rate of NK1R
endocytosis was quantified with 125I-SP (27). Cells were
incubated in Hanks' balanced salt solution containing 50 pM 125I-SP, 0.1% bovine serum albumin for 60 min at 4 °C. They were washed and incubated at 37 °C for 0-10
min. Cells were washed with ice-cold PBS and incubated in 250 µl of
ice-cold 0.2 M acetic acid containing 500 mM
NaCl (pH 2.5) on ice for 5 min to separate acid-labile (cell surface)
from acid-resistant (internalized) label. Nonspecific binding was
measured in the presence of 1 µM SP and was subtracted to
give specific binding. Observations were in triplicate in
n > 3 experiments. KNRK cells expressing empty vector
without NK1R insert do not bind or take up 125I-SP
(50).
Measurement of
[Ca2+]i--
[Ca2+]i
was measured in populations of transfected cells using Fura-2/AM (11,
27). Fluorescence was measured at 340- and 380-nm excitation and 510-nm
emission, and the results were expressed as the ratio of the
fluorescence at the two excitation wavelengths, which is proportional
to the [Ca2+]i. Cells were exposed once to SP to
generate concentration-response curves. For desensitization
experiments, cells were exposed to SP or vehicle (control) for 2 min,
washed, and exposed again to SP 5 min after the first exposure. To
examine resensitization, cells were incubated with SP or vehicle
(control) for 10 min, washed, and challenged with SP 0-180 min after
washing. All observations were in n > 3 experiments.
KNRK cells expressing empty vector without the NK1R insert do not
mobilize [Ca2+]i in response to SP (50).
MAP Kinase Assays--
SP-induced phosphorylation of ERK1/2 was
determined by Western blotting (15, 16). Similar results were obtained
in kinase assays using myelin basic protein as a substrate (data not
shown). Cells were maintained serum-free medium overnight and incubated with 10 nM SP for 0-30 min at 37 °C. Cells were lysed
in boiling 20 mM Tris-HCl, pH 8, 10 mM EDTA,
0.3% SDS, 67 mM DTT. Lysates (20 µg of protein) were
analyzed by 12% SDS-PAGE and transferred to polyvinylidene difluoride
membranes. Membranes were incubated with pERK1/2 (pERK) antibody
(1:1000, overnight, 4 °C), followed by goat anti-mouse IgG
conjugated to horseradish peroxidase (1:30,000, 1 h, room
temperature). Proteins were visualized by ECL. Blots were stripped and
reprobed with antibody to total ERK1/2 to ensure that equal levels of
ERK1/2 were present at each time point. The intensities of the combined
ERK1 and ERK2 bands for phosphorylated and total proteins were
determined by histogram analysis (mean density × number pixels)
(15, 16). Intensities of the pERK bands were normalized to total ERKs
to correct for any differences in loading. Results are expressed as the
-fold increase in pERK1/2 relative to unstimulated cells. All
observations were in n = 4 experiments.
NK1R Trafficking in Cell Lines--
Cells were incubated with
10-100 nM Alexa594-SP for 60 min at 4 °C (for
equilibrium binding), washed at 4 °C, and either fixed immediately
or incubated in SP-free medium at 37 °C for 2.5-10 min (for
trafficking to proceed) (11, 18). Cells were fixed with 4%
paraformaldehyde in 100 mM PBS, pH 7.4, for 20 min at 4 °C. NK1R was localized using Alexa-SP or by immunofluorescence using the M2 FLAG antibody (10 µg/ml, 18 h, 4 °C) or an
antibody to the C terminus of the rat NK1R (1:1,000, 18 h,
4 °C). Rab5a was localized using EGFP, and dynamin was localized by
immunofluorescence using the HA antibody (4 µg/ml, 18 h,
4 °C) (11, 18). Fluorescent SP does not bind to untransfected KNRK
cells (45).
NK1R Trafficking in Enteric Neurons--
Organotypic cultures
were prepared from the rat ileum (Harlan Sprague-Dawley, male, 200-250
g, Harlan Laboratories, San Diego, CA) as previously described for
guinea pigs (48). Excised segments of distal were opened along the
mesentery, pinned flat, and incubated in Krebs solution containing 100 nM SP for 1 h at 4 °C. Specimens were either
immediately fixed or incubated in SP-free medium for 20 min at 37 °C
(48). Specimens were fixed in 4% paraformaldehyde for 2 h and
washed, and whole mounts of the longitudinal muscle with attached
myenteric plexus were prepared. Tissues were processed for sequential
immunofluorescence using a C-terminally directed rabbit antibody to
NK1R (1:500, 48 h, 4 °C) and a monoclonal dynamin antibody
(1:50, 48 h, 4 °C), followed by secondary antibodies labeled with contrasting fluorophores. The sequential double labeling procedure was chosen because preliminary experiments showed that mixing
the NK1R and dynamin antibodies did not give clear staining of
individual antigens. Myenteric neurons from the small intestine of
newborn male guinea pigs (Duncan-Hartley; Simonsen, Gilroy, CA) were
dispersed and cultured exactly as described (30, 31). Neurons were
studied at days 7-14 of culture. Neurons were incubated with 100 nM Alexa594-SP or
Alexa594-[Sar9,MetO211]SP for 120 min at 4 °C, washed at 4 °C, and either fixed immediately or
incubated in SP-free medium at 37 °C for 5-30 min (30, 31). Neurons
were fixed with 4% paraformaldehyde for 20 min at 4 °C. NK1R was
detected using Alexa-SP, and Rab5a was localized by immunofluorescence with a polyclonal antibody (1:50, 2 h, 4 °C) (30,
31).
Microscopy--
Specimens were observed using a Zeiss Axiovert
100 microscope (Carl Zeiss Inc., Thornwood, NY) with an MRC 1000 confocal microscope (Bio-Rad) or a Zeiss 410 confocal microscope (30,
48). Images were collected at 0.5-µm intervals using a × 100 PlanApo 1.4 NA objective and a zoom of 1-2 and were processed using
Adobe Photoshop (Adobe Systems, Mountain View, CA).
Statistical Analysis--
Results are expressed as means ± S.E. Differences between values were analyzed by one-way
analysis of variance and the Student-Newman-Keul's test, with
p < 0.05 considered to be significant.
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RESULTS |
Characterization of Cell Lines Expressing NK1R,
Dynamin, and Rab5a--
We selected KNRK cells, since they regulate
NK1R signaling and trafficking similarly to neurons that naturally
express the NK1R (11, 18, 27, 30, 31). We transiently transfected KNRK-FLAGNK1R cells with HA-dynamin-pIRES-GFP or HA-dynamin
K44E-pIRES-GFP and sorted EGFP-expressing cells (Fig.
1A). Western blotting
identified an intensely immunoreactive band corresponding to dynamin in
cells expressing dynamin and dynamin K44E, which was far more intense than that observed in nontransfected cells (Fig. 1B). NK1R
was detected at the cell surface in close proximity to dynamin and dynamin K44E, which were also detected in the cytosol (Fig.
1C). We stably expressed the FLAGNK1R in a
hygromycin-resistant vector and Rab5a-GFP or Rab5aS34N-GFP in a
neomycin-resistant vector. We used KNRK-NK1R+Rab5a clone 10 and
KNRK-NK1R+Rab5aS34N clone 3 in all experiments. Flow cytometry revealed
a single population of cells highly expressing NK1R plus Rab5a or
Rab5aS34N (Fig. 2A). Western
blotting with a Rab5a antibody detected endogenous Rab5a (~30 kDa) in
all cell lines and also detected Rab5a-GFP (~55 kDa) corresponding to
Rab5a (~30 kDa) plus EGFP (~27 kDa) in transfected cells (Fig.
2B, left panel). The EGFP antibody detected only a band at ~55 kDa cells expressing Rab5a-GFP or Rab5aS34N-GFP (Fig. 2B, right panel).
Immunoreactive NK1R was localized to the plasma membrane, Rab5a was
detected in superficial and perinuclear vesicles, and Rab5aS34N was
mostly present in the cytosol (Fig. 2C). To verify that
cells expressed functional NK1R, we measured SP-induced
Ca2+ mobilization. In all cells, SP stimulated a prompt
increase in [Ca2+]i (not shown, but see Fig. 8)
with EC50 values similar to that measured in cells
expressing NK1R alone (~0.6 nM).

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Fig. 1.
Characterization of cells expressing FLAGNK1R
plus HA-dynamin or HA-dynamin K44E. KNRK-FLAGNK1R cells were
transiently transfected with HA-dynamin or HA-dynamin K44E in the
pIRES-GFP vector. A, analysis by flow cytometry confirmed
the high efficiency of transfection. B, Western blotting of
enriched cells using a dynamin antibody revealed high expression of
dynamin in transfected compared with untransfected cells. C,
confocal microscopy showed colocalization of NK1R (detected with FLAG
antibody) with dynamin and dynamin K44E (detected with HA.11 antibody)
at the plasma membrane (arrowheads). Scale
bar, 10 µm.
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Fig. 2.
Characterization of cells expressing FLAGNK1R
plus Rab5a or Rab5aS34N. KNRK cells were stably transfected with
FLAGNK1R plus Rab5a-GFP or Rab5aS34N-GFP. A, analysis by
flow cytometry confirmed coexpression of NK1R (detected with FLAG
antibody) and Rab5a or Rab5aS34N (detected with EGFP). B,
Western blotting using Rab5a or EGFP antibodies revealed high
expression of Rab5a-GFP and Rab5aS34N-GFP chimeras of the predicted
mass. C, confocal microscopy showed localization of NK1R
(detected with FLAG antibody) at the cell surface
(arrowheads), Rab5a-GFP in endosomes (arrows),
and Rab5aS34N-GFP throughout the cytosol (arrows).
Scale bar, 10 µm.
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Dynamin and Rab5a Mediate Endocytosis of
125I-SP--
To quantitatively evaluate the importance of
dynamin and Rab5a in NK1R endocytosis, we measured internalization of
125I-SP. Cells were incubated with 125I-SP for
60 min at 4 °C, washed, and incubated for 0-10 min at 37 °C, and
surface and internalized label were separated by an acid wash. In
KNRK-NK1R+DYN cells, 92 ± 8% of specifically bound SP was at the
cell surface, and 8 ± 1% was internalized after 60 min at
4 °C (Fig. 3A). Warming to
37 °C resulted in rapid internalization of 125I-SP that
was maximal within 10 min (28 ± 7% internalized at 2.5 min,
81 ± 7% at 5 min, and 90 ± 5% at 10 min) and was mirrored by a concomitant decline in surface 125I-SP. Similar
results were obtained in cells expressing NK1R alone (4 ± 2%
internalized at 0 min, 36 ± 5% 2.5 min, 77 ± 2% 5 min, 82 ± 1% 10 min). Expression of dynamin K44E did not affect SP binding at 4 °C but retarded endocytosis after warming (Fig.
3A). Inhibition was greatest at 5 min, when 82 ± 6%
of specifically bound 125I-SP was internalized in
KNRK-NK1R+DYN cells and only 45 ± 6% in KNRK-NK1R+DYNK44E cells
(p < 0.05), representing an inhibition of endocytosis
of 45%. In KNRK-NK1R+Rab5a cells, 95 ± 1% of specifically bound
125I-SP was at the surface, and 5 ± 1% was
internalized after 60 min at 4 °C (Fig. 3B). Warming to
37 °C resulted in rapid internalization of 125I-SP that
was maximal within 10 min (37 ± 3% internalized at 2.5 min,
81 ± 1% 5 min, 84 ± 1% 10 min). Expression of Rab5aS34N
did not affect binding at 4 °C but reduced endocytosis after warming (Fig. 3B). Inhibition was greatest at 10 min, when 84 ± 1% of specifically bound 125I-SP was internalized in
KNRK-NK1R+Rab5a cells and only 56 ± 4% in KNRK-NK1R+Rab5aS34N
cells (p < 0.05, 32% inhibition). The inhibitory effects of dynamin K44E and Rab5aS34N diminished at 30 min (not shown).
Thus, both dynamin and Rab5a mediate the early stages of SP-induced
endocytosis of the NK1R.

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Fig. 3.
Endocytosis of
125I-SP in KNRK-FLAGNK1R expressing HA-dynamin or
HA-dynamin K44E in the pIRES-EGFP vector (A) and in
KNRK-FLAGNK1R+Rab5a or KNRK-FLAGNK1R+Rab5aS34N cells
(B). Expression of dominant negative dynamin K44E
and Rab5aS34N strongly inhibited endocytosis. *, p < 0.05 compared with wild-type cells, n = 3 or 4 experiments in triplicate.
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Dynamin Mediates Translocation of the Alexa-SP from the Cell
Surface to Superficial Early Endosomes--
To identify the steps of
endosome formation and trafficking that are mediated by dynamin, we
localized the NK1R using Alexa594-SP with dynamin or dynamin K44E by
immunofluorescence. Cells were incubated with Alexa594-SP for 60 min at
4 °C, washed, and incubated at 37 °C for 0-10 min. In cells
expressing wild-type dynamin (KNRK-NK1R+DYN) after 60 min at 4 °C,
dynamin was detected at the plasma membrane and in the cytosol, and
Alexa594-SP and thus the NK1R were at the cell surface (Fig.
4, arrowheads). After 10 min
at 37 °C, Alexa594-SP was detected in superficial and then
perinuclear endosomes and dynamin remained at the plasma membrane (Fig.
4). In cells expressing dominant negative dynamin (KNRK-NK1R+DYNK44E)
at 4 °C, dynamin K44E was detected in the cytosol at the plasma
membrane, and Alexa594-SP was at the cell surface (Fig.
5). However, after 10 min at 37 °C,
Alexa594-SP was retained at the cell surface in cells expressing
dynamin K44E (Fig. 5, arrowheads). This result is in marked
contrast to that observed in cells expressing wild-type dynamin (Fig.
4) or to untransfected cells (Fig. 5, asterisk). Retention
at the cell surface was even observed after 30 min (not shown). Thus,
dynamin mediates formation of early endosomes containing the NK1R.

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Fig. 4.
Localization of Alexa594-SP and
wild-type dynamin. KNRK-FLAGNK1R cells were transiently
transfected with HA-dynamin. Cells were incubated with Alexa594-SP for
60 min at 4 °C, washed, and incubated at 37 °C for 0 or 10 min.
Dynamin was localized by immunofluorescence using the HA.11
antibody. The same cells are shown in each row, and the
images in the right panels are
superimpositions of the images in the same
row. After 0 min at 37 °C, Alexa594-SP was at the cell
surface (arrowheads), and dynamin was in the cytoplasm and
at the plasma membrane (arrowheads). After 10 min,
Alexa594-SP was detected in perinuclear endosomes (arrows),
and dynamin remained at the plasma membrane (arrowheads).
Scale bar, 10 µm. Results shown are
representative of three experiments.
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Fig. 5.
Localization of Alexa594-SP and
dynamin K44E. KNRK-FLAGNK1R cells were transiently transfected
with HA-dynamin K44E. Cells were incubated with Alexa594-SP for 60 min
at 4 °C, washed, and incubated at 37 °C for 0 or 10 min. Dynamin
K44E was localized by immunofluorescence using the HA.11 antibody. The
same cells are shown in each row, and the images
in the right panels are superimpositions of the
images in the same row. After 0 min at 37 °C,
Alexa594-SP was at the cell surface (arrowheads), and
dynamin was in the cytoplasm and at the plasma membrane
(arrowheads). After 10 min at 37 °C, in cells expressing
dynamin K44E, Alexa594-SP was found at the cell surface
(arrowheads). In contrast, Alexa-SP was detected in
perinuclear endosomes in untransfected (asterisks,
arrows). Dynamin K44E remained at the cell surface
(arrowheads). Scale bar, 10 µm.
Results shown are representative of three experiments.
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Rab5a Mediates Translocation of the Alexa-SP from the Cell Surface
to Superficial and Perinuclear Early Endosomes--
We similarly
localized the NK1R and Rab5a. In cells expressing wild-type Rab5a
(KNRK-NK1R-Rab5a) after 60 min at 4 °C, Rab5a-GFP was detected in
multiple prominent vesicles and weakly at the plasma membrane, and
Alexa594-SP was confined to the cell surface (Fig.
6). After 2.5 min at 37 °C,
Alexa594-SP was localized to superficial vesicles, which also contained
Rab5a-GFP (Fig. 6, arrows). After 5 and 10 min, Alexa594-SP
was also detected in perinuclear vesicles containing Rab5a-GFP. We have
previously reported that vesicles containing SP and the NK1R also
contain the transferrin receptor and are thus early endosomes (11, 18). Similarly, Alexa594-SP underwent rapid endocytosis in cells not expressing Rab5a-GFP (Fig. 6, asterisk). In cells expressing
dominant negative Rab5a (KNRK-NK1R-Rab5aS34N) at 4 °C, Rab5aS34N-GFP
was in the cytosol and Alexa594-SP was confined to the cell surface (Fig. 7). After 2.5 min at 37 °C,
Alexa594-SP was retained at the cell surface in KNRK-NK1R-Rab5aS34N
cells (Fig. 7, arrowheads). In marked contrast, in
KNRK-NK1R-Rab5a cells (Fig. 6) and in untransfected cells (Fig. 7,
asterisk) Alexa594-SP was detected in superficial endosomes
at 2.5 min. After 5 and 10 min in KNRK-NK1R-Rab5aS34N cells,
Alexa594-SP was present at the cell surface (Fig. 7,
arrowheads) and in superficial endosomes that did not
proceed to a perinuclear location (Fig. 7, yellow
arrows). In contrast, in KNRK-NK1R-Rab5a cells (Fig. 6) and
untransfected cells (Fig. 7, asterisk) Alexa594-SP was found
in perinuclear endosomes at 5 and 10 min. Thus, Rab5a mediates
formation of early endosomes and their translocation to a perinuclear
location.

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Fig. 6.
Localization of Alexa594-SP and wild-type
Rab5a-GFP. KNRK-FLAGNK1R cells were transiently transfected with
Rab5a-GFP. Cells were incubated with Alexa594-SP for 60 min at 4 °C,
washed, and incubated at 37 °C for 0-10 min. The same cells are
shown in each row, and the images in the
right panels are superimpositions of the images
in the same row. After 0 min at 37 °C,
Alexa594-SP was at the cell surface (arrowheads). After 2.5, 5, and 10 min, Alexa594-SP was detected in superficial and then
perinuclear endosomes containing Rab5a-GFP (arrows).
Alexa-594-SP was similarly internalized in untransfected cells
(asterisks). Scale bar, 10 µm.
Results shown are representative of three experiments.
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Fig. 7.
Localization of Alexa594-SP and
Rab5aS34N-GFP. KNRK-FLAGNK1R cells were transiently transfected
with Rab5aS34N-GFP. Cells were incubated with Alexa594-SP for 60 min at
4 °C, washed, and incubated at 37 °C for 0-10 min. The same
cells are shown in each row, and the images in
the right panels are superimpositions of the
images in the same row. After 0 min at 37 °C, Alexa594-SP
was at the cell surface (arrowheads). In cells expressing
Rab5aS34N-GFP, Alexa594-SP remained at the cell surface at 2.5 min
(arrowheads). At 5 and 10 min, Alexa594-SP was found at the
cell surface (arrowheads) and in superficial endosomes
(yellow arrows) that did not proceed to a
perinuclear location. In contrast, in untransfected cells, Alexa594-SP
rapidly internalized into superficial and then perinuclear endosomes
(asterisks, white arrows).
Scale bar, 10 µm. Results shown are
representative of three experiments.
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Dynamin and Rab5a-mediated Endocytosis of the NK1R Are Not Required
for Desensitization of SP-induced Ca2+
Mobilization--
Exposure of KNRK-NK1R cells co-expressing dynamin or
dynamin K44E (Fig. 8A) and
Rab5a or Rab5aS34N (Fig. 8B) to 10 nM SP for 2 min caused a prompt and transient increase in
[Ca2+]i, confirming expression of functional
receptors. When cells were washed and exposed again to 10 nM SP 5 min after the first challenge, the response was
strongly desensitized in all cell lines (Fig. 8, A and
B). We have previously reported a similar desensitization in
cells expressing NK1R alone and shown that desensitization is not due
to depletion of intracellular Ca2+ stores (27, 28).
Desensitization occurred at a time when the NK1R was internalized in
cells expressing wild-type dynamin and Rab5a but was at the cell
surface in cells expressing dominant negative dynamin and Rab5a. Thus,
dynamin- and Rab5a-mediated endocytosis are not required for
desensitization of the NK1R.

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Fig. 8.
Desensitization of SP-induced
Ca2+ mobilization in KNRK-FLAGNK1R expressing HA-dynamin or
HA-dynamin K44E in the pIRES-EGFP vector (A) and in
KNRK-FLAGNK1R+Rab5a or KNRK-FLAGNK1R+Rab5aS34N cells
(B). Cells were exposed to 10 nM SP
or carrier (control) for 2 min, washed, and challenged with 10 nM SP 5 min later. In all cell lines, SP strongly
desensitized the NK1R. Shown are representative traces of four
observations.
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Dynamin- and Rab5a-mediated Endocytosis of the NK1R Are Required
for Resensitization of SP-induced Ca2+
Mobilization--
To examine resensitization, cells were incubated
with 10 nM SP or vehicle for 10 min, washed, and challenged
with 10 nM SP at 0-180 min after washing. In cells
expressing FLAGNK1R alone, exposure to 10 nM SP for 10 min
strongly desensitized SP-induced Ca2+ mobilization, which
gradually resensitized when the interval between SP challenges
increased beyond 10 min, such that complete resensitization occurred
after 180 min (Fig. 9A). A
similar degree of desensitization was observed in cells expressing
wild-type or dominant negative dynamin or Rab5a (Fig. 9B).
Resensitization was almost complete after 180 min in cells expressing
wild-type dynamin (91 ± 23% resensitization) or Rab5a (79 ± 34% resensitization) (Fig. 9B). In marked contrast,
after 180 min in cells expressing dynamin K44E or Rab5aS34N, responses
to a second challenge with SP were resensitized by only 36 ± 12%
and 12 ± 5%, respectively (p < 0.05) (Fig.
9B). Compared with cells expressing wild-type dynamin and
Rab5a, dynamin K44E inhibited resensitization by 60%, and dominant
negative Rab5aS34N inhibited resensitization by 85%. Thus, dynamin-
and Rab5a-mediated trafficking of the NK1R are required for
resensitization of responses to SP.

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Fig. 9.
Resensitization of SP-induced
Ca2+ mobilization in KNRK cells expressing FLAGNK1R alone
and in KNRK-FLAGNK1R expressing HA-dynamin or HA-dynamin K44E in the
pIRES-EGFP vector and in KNRK-FLAGNK1R+Rab5a or KNRK-FLAGNK1R+Rab5aS34N
cells. Cells were exposed to 10 nM SP or vehicle
(control) for 10 min, washed, and challenged with 10 nM SP
0-180 min later. A, the time course in cells expressing
NK1R; B, response at 0 and 180 min in cells expressing
wild-type and dominant negative dynamin and Rab5a. Note that expression
of dynamin K44E and Rab5aS34N had no effect on desensitization at 0 min
but strongly inhibit resensitization at 180 min. *, p < 0.05 compared with wild-type cells; n = 3 or 4 experiments in triplicate.
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Dynamin but Not Rab5a-mediated Endocytosis Is Required for
Mitogenic Signaling of the NK1R--
To determine the role of dynamin
and Rab5a in SP activation of the MAP kinase cascade, we compared
SP-induced ERK1/2 activation in cell lines expressing wild-type and
dominant negative dynamin or Rab5a. Cells were incubated with SP for
0-30 min, and activation was determined by Western blotting using
antibodies to pERK1/2 (activated) and total ERK1/2. Under basal
conditions, there was a low level of phosphorylation of ERK1/2 in all
cell lines. Densitometric analysis of the pERK1/2 and total ERK1/2
indicated that the basal level of activation was similar in all cells
(pERK1/2 versus total ERK1/2 ratios: KNRK-NK1R+DYN, 0.6 ± 0.1; KNRK-NK1R+DYNK44EK, 0.5 ± 0.1; NRK-NK1R+Rab5a, 0.6 ± 0.1; KNRK-NK1R+Rab5aS34N, 0.5 ± 0.1). In cells expressing
wild-type dynamin, SP stimulated a prompt increase in ERK1/2
phosphorylation that was maximal at 5 min (2.4-fold over basal) and
remained elevated in the continued presence of SP after 30 min (Fig.
10A). We have previously
reported similar results in KNRK cells expressing the NK1R alone and
endothelial cells that naturally express the NK1R (16). Expression of
EGFP alone did not alter this response (not shown). Expression of
dynamin K44E strongly inhibited SP-induced phosphorylation of ERK1/2
(1.1-fold over basal at 5 min, p < 0.05). In cells
expressing wild-type Rab5a, SP stimulated a rapid phosphorylation of
ERK1/2 that was maximal after 2.5 min (2-fold over basal) and remained
elevated for 30 min (Fig. 10B). Expression of Rab5aS34N had
no effect on SP-induced phosphorylation of ERK1/2 (2-fold over basal at
2.5 min). Thus, dynamin- but not Rab5a-mediated trafficking of the NK1R
is required for mitogenic signaling.

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Fig. 10.
SP-induced activation of ERK1/2. Cells
expressing the NK1R with wild-type or dominant negative dynamin
(A) or Rab5a (B) were incubated with 10 nM SP for the indicated times. Cells were analyzed by
Western blotting using antibodies to pERK1/2 or total ERK1/2, for
standardization. The upper panels show
representative Western blots, and lower panels
show the -fold increase over basal phosphorylation. *,
p < 0.05 compared with wild-type cells,
n = 4 experiments.
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Alexa594-SP Colocalizes with Rab5a in Endosomes in Enteric Neurons
Expressing the NK1R--
To confirm a potential role of dynamin and
Rab5a in nontransfected cells, we studied myenteric neurons that
naturally express the NK1R (30, 31, 46). We used rat neurons for
studies of dynamin and guinea pig neurons for studies of Rab5a due to
limitations in antibody specificity. Organotypic cultures of rat ileum
were incubated with SP for 60 min at 4 °C, washed, and incubated for 0-20 min at 37 °C. The NK1R and dynamin were localized by
immunofluorescence. At 4 °C, the NK1R was detected at the cell
surface in the soma of a subpopulation of neurons (Fig.
11). Dynamin was detected in NK1R-positive neurons and in most other neurons, where it was predominantly cytosolic and also in close proximity to the plasma membrane. After 20 min at 37 °C, the NK1R was detected in endosomes, whereas the distribution of dynamin was unchanged. Cultures of guinea
pig neurons were incubated with Alexa594-SP or
Alexa594-[Sar9,MetO211]SP
(similar results were obtained with both peptides) for 120 min at
4 °C, washed, and incubated for 0-30 at 37 °C. Rab5a was localized by immunofluorescence. After 0-2 min at 37 °C,
Alexa594-SP and, thus, the NK1R were detected at the cell surface, and
Rab5a was in endosomes (Fig. 12). After
5-10 min at 37 °C, Alexa594-SP was colocalized with Rab5a in
endosomes in the soma and neurites. We have previously shown that these
vesicles contain the transferrin receptor and are thus early endosomes
(30). After 30 min, Alexa594-SP was present in perinuclear endosomes
that did not contain Rab5a (not shown). Thus, myenteric neurons
expressing the NK1R also express dynamin and Rab5a, and these proteins
are appropriately localized within the cell to regulate trafficking of
the NK1R.

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Fig. 11.
Localization of NK1R and dynamin in rat
myenteric neurons. Neurons were incubated with SP for 60 min at
4 °C, washed, and incubated at 37 °C for 0 or 20 min. NK1R and
dynamin were detected by immunofluorescence. The same neurons are shown
in each row, and the images in the
right panels are superimpositions of the
images in the same row. At 0 min, the NK1R was at
the cell surface, and dynamin was in the cytosol close to the plasma
membrane. After 20 min, NK1R was detected in endosomes
(arrows), and the distribution of dynamin was unchanged.
Scale bar, 10 µm.
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Fig. 12.
Localization of Alexa594-SP or and Rab5a in
guinea pig myenteric neurons. Neurons were incubated with
Alexa594-SP for 120 min at 4 °C, washed, and incubated at 37 °C
for 2 or 10 min. Rab5a was detected by immunofluorescence using a
fluorescein isothiocyanate-conjugated secondary antibody. The same
neurons are shown in each row, and the images in
the right panels are superimpositions of the
images in the same row. At 2 min, the NK1R was at
the cell surface (arrowheads), and Alexa-SP was in
endosomes. After 10 min, Alexa594-SP was in endosomes containing Rab5a
(arrows). Scale bar, 5 µm.
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DISCUSSION |
We found that expression of dominant mutants of dynamin 1 and
Rab5a retarded SP-induced endocytosis of the NK1R. Dominant negative
Rab5a also impeded the intracellular trafficking of endosomes containing the NK1R from a superficial to a perinuclear region. These
mutants strongly inhibited resensitization of SP-induced Ca2+ mobilization but did not affect desensitization.
Whereas dominant negative dynamin almost abolished SP-stimulated
activation of ERK1/2, dominant negative Rab5a had no effect. Thus, our
results indicate that dynamin and Rab5a are required for the formation of endosomes containing the NK1R and that Rab5a mediates their translocation to a perinuclear region. Dynamin and
Rab5a-dependent trafficking of the NK1R is essential for
resensitization of responses to SP, and dynamin-mediated endosome
formation is required for coupling of the NK1R to the MAP kinase
pathway. Moreover, dynamin and Rab5a are appropriately localized in
neurons expressing the NK1R to suggest that they participate in the
physiological regulation of NK1R trafficking and signaling. To our
knowledge, this study is the first to demonstrate a role for dynamin
and Rab5a in trafficking and signaling of the NK1R and the first report
of the relative roles of dynamin and Rab5a in mitogenic signaling of a
GPCR.
Molecular Mechanisms of SP-induced Endocytosis and Intracellular
Trafficking of the NK1R--
Dynamin was detected at or near the
plasma membrane under basal conditions, and dynamin K44E inhibited
endocytosis of 125I-SP by ~50% at early time points (5 min) and caused retention of most Alexa-SP at the cell surface for
prolonged periods (up to 30 min). The larger effect of dynamin K44E on
endocytosis of Alexa-SP than on endocytosis of 125I-SP,
suggests that dynamin K44E permits 125I-SP to be
sequestered into a compartment at or near the plasma membrane, where it
is resistant to removal by the acid wash that was used to separate cell
surface from internalized peptide. However, the consensus of our
results is that dynamin plays an important role in SP-induced
endocytosis of the NK1R, and our findings support the hypothesis that
dynamin mediates the final stages of endosome formation from
clathrin-coated pits (32, 33). The observation that SP stimulates
endocytosis of the NK1R by a clathrin-mediated mechanism in KNRK cells
and enteric neurons supports a role for dynamin in endosome formation
(18, 30). In support of our results, dynamin mediates endocytosis of
receptors that constitutively internalize, such as the transferrin
receptor (34), as well as GPCRs that internalize in response to agonist
binding, including the
2-AR;
-opioid; muscarinic m1,
m3, and m4; and dopamine D1 receptors (12, 35, 37-39). However,
dynamin is not required for endocytosis of angiotensin II type 1A,
muscarinic m2, dopamine D2, and
2B-adrenergic receptors
(12, 37, 39, 40). The mechanisms of dynamin-independent endocytosis and
the receptor domains that specify dynamin dependence remain to be determined.
Rab5a prominently colocalized with the internalized NK1R in superficial
and perinuclear endosomes and is thus appropriately localized within
cells to regulate NK1R trafficking. Rab5aS34N inhibited endocytosis of
125I-SP by ~35% and retarded endocytosis of Alexa-SP,
supporting a role for Rab5a SP-induced endocytosis of the NK1R. In
support of our results, Rab5a mediates the formation of endosomes
containing the transferrin receptor (41-43), dopamine D2 receptor
(38), and the
2-AR (44). Rab5aS34N also caused retention
of the NK1R in superficial endosomes, indicating a requirement of Rab5a
for translocation of endosomes from a superficial to a perinuclear region. Dominant negative Rab5a also impedes the kinetics of membrane trafficking in the endocytic pathway (41) and similarly causes retention of the
2-AR in endosomes close to the plasma
membrane (44).
Although expression of dynamin K44E and Rab5aS34N markedly retarded SP
endocytosis of the NK1R, they did not abolish this trafficking.
Explanations include the possibility that the mutants were not
expressed at high enough levels to completely inhibit the endogenous
proteins or that additional mechanisms also contribute to trafficking.
-Arrestins couple certain GPCRs to clathrin, including
2-AR, m2 muscarinic receptor, protease-activated
receptor 2, and the NK1R (9-11, 23, 51). To interact with
-arrestins, receptors must undergo phosphorylation, and G-protein
receptor kinases 2 and 3 phosphorylate the NK1R in reconstituted
systems and in cell lines (52-55). Dynamin acts downstream from
-arrestins, by participating in endosome formation at
clathrin-coated pits (32, 33). Rab5a is likely to participate in both
the formation of these endosomes and their translocation to a
perinuclear location (56, 57).
Role of SP-induced Endocytosis and Intracellular Trafficking of the
NK1R for Signal Transduction--
Agonist-induced trafficking of GPCRs
and associated proteins regulates cellular responses (1, 2). SP induces
translocation of
-arrestins to the plasma membrane, where they
interact with the NK1R to mediate desensitization and endocytosis (11,
31). Endocytosis could contribute to desensitization by depleting the cell surface of receptors. However, dynamin K44E and Rab5aS34N, which
inhibited NK1R endocytosis, did not affect desensitization of
SP-induced Ca2+ mobilization, indicating that NK1R
endocytosis is not the main mechanism of desensitization. In support of
these findings, inhibition of NK1R endocytosis by dominant negative
-arrestin-(319-418) or with drugs does not prevent desensitization
(11, 20, 27). Thus, uncoupling of the NK1R from G-proteins is the main
mechanism of desensitization. In a similar manner, uncoupling is the
principal mechanism of desensitization of the
2-AR (1).
However, down-regulation of the
-opioid receptor following prolonged
activation requires endocytosis by dynamin,
-arrestin and
Rab5a-dependent mechanisms (24).
Dynamin K44E and Rab5aS34N markedly inhibited resensitization of
SP-induced Ca2+ mobilization by ~60-85%. These results
indicate that endocytosis and trafficking of the NK1R is necessary for
recovery of responses to SP. In support of these findings,
pharmacological inhibition of endocytosis and recycling and the NK1R
and
2-AR disrupts their resensitization (19, 20), and
Rab5aS34N also inhibits resensitization of
2-AR (44).
Thus, resensitization of GPCRs requires endocytosis, dissociation from
the receptor from ligand in acidified endosomes, receptor
dephosphorylation and dissociation from
-arrestins, and recycling.
Although agonist-stimulated endocytosis of some GPCRs is necessary for
activation of the MAP kinase cascade, little is known about the
relative contributions of
-arrestin, dynamin and Rab5a to this
process. We found that dynamin K44E, but not Rab5aS34N, abolished
SP-induced activation of ERK1/2. We have previously reported that
dominant negative
-arrestin-(319-418) similarly inhibits
this process (16). In support of our results,
-arrestin- and
dynamin-dependent endocytosis is also required for coupling the
2-AR,
-opioid receptor, m1 muscarinic receptor,
and proteinase-activated receptor-2 to the MAP kinase pathway (14, 15,
36, 58, 59).
-Arrestins serve as scaffolds that recruit and organize components of the MAP kinase cascade including Src, in the case of the NK1R and
2-AR (14, 16), and Raf-1 in the
case of the proteinase-activated receptor-2, where this interaction
also determines the location and specificity of activated ERK1/2 (15).
We found that inhibition of the proximal steps of endocytosis, by
expression of dominant negative mutants of
-arrestin and dynamin,
inhibited SP-induced ERK1/2 activation, whereas disruption of Rab5a,
which acts more distally, was without effect. Thus, for the same GPCR, inhibition of distinct steps of endocytosis and trafficking has different effects on coupling to the MAP kinase cascade. However, expression of dominant negative mutants of
-arrestin and dynamin does not affect coupling of the CXCR2,
-opioid, or
2-adrenergic receptors to ERK1/2 (60-62). Dynamin may
have independent roles in GPCR internalization and MAP kinase
activation, since dominant negative dynamin inhibits phorbol
ester-stimulated MAP kinase activation, which is independent of
receptor endocytosis and transactivation (63).
Physiological Relevance of NK1R Trafficking--
An understanding
of the molecular mechanisms that regulate signaling by SP in the
nervous system is of great interest, since the NK1R plays a major role
in pain, depression, neurogenic inflammation, and gastrointestinal
motility and secretion (25, 26). We found that myenteric neurons
expressing the NK1R also express dynamin and Rab5a and that dynamin and
Rab5a are appropriately located to regulate endocytosis and trafficking
of the NK1R in the soma. SP induces membrane translocation of G-protein
receptor kinases 2 and 3 and
-arrestin 1/2 in these neurons, and
disruption of the formation of clathrin-coated pits inhibits receptor
endocytosis (30, 31). Thus, it is likely that clathrin,
-arrestins,
dynamin, and Rab5a mediate SP-induced endocytosis of the NK1R in
neurons, as they do in transfected KNRK cells. Defects in these
regulatory mechanisms may result in unregulated signaling and disease.
Deletion of neutral endopeptidase EC 3.4.24.11, which degrades SP at the cell surface and thereby terminates signaling, results in exaggerated inflammatory effects of SP (64, 65). Alterations in the
expression of other regulatory proteins, such as
-arrestins, dynamin
and Rab5a, may also result in uncontrolled signaling and disease.