Báo cáo khoa học: Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of b-arrestin 1 recruitment potx

In conclusion, we demonstrate that CRFR1 internalization is independent of phosphory-lation sites in the C-terminal tail and third intracellular loop, and the degree of b-arrestin 1 recr

Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of b-arrestin 1 recruitment

Trine N Rasmussen1,3, Ivana Novak3and Søren M Nielsen2

1

Department of Molecular Biology and2Department of Molecular Pharmacology, H Lundbeck A/S, Valby, Denmark;

3

August Krogh Institute, University of Copenhagen, Denmark

The corticotropin releasing factor receptor 1 (CRFR1)

belongs to the superfamily of G-protein coupled receptors

Though CRF is involved in the aetiology of several

stress-related disorders, including depression and anxiety, details of

CRFR1 regulation such as internalization remain

unchar-acterized In the present study, agonist-induced

internal-ization of CRFR1 in HEK293 cells was visualized by

confocal microcopy and quantified using the radioligand

125I-labelled sauvagine Recruitment of b-arrestin 1 in

re-sponse to receptor activation was demonstrated by confocal

microscopy The extent of125I-labelled sauvagine stimulated

internalization was significantly impaired by sucrose,

indi-cating the involvement of clathrin-coated pits No effect on

the extent of internalization was observed in the presence of

the second messenger dependent kinase inhibitors H-89 and

staurosporine, indicating that cAMP-dependent protein kinase and protein kinase C are not prerequisites for CRFR1 internalization Surprisingly, deletion of all putative phos-phorylation sites in the C-terminal tail, as well as a cluster of putative phosporylation sites in the third intracellular loop, did not affect receptor internalization However, these mutations almost abolished the recruitment of b-arrestin 1 following receptor activation In conclusion, we demonstrate that CRFR1 internalization is independent of phosphory-lation sites in the C-terminal tail and third intracellular loop, and the degree of b-arrestin 1 recruitment

Keywords: b-arrestin 1; CRFR1; GPCR; receptor internal-ization

Corticotropin releasing factor (CRF) is a 41-residue

neuro-peptide first isolated in 1981 [1] as the main stimulator of the

release of adenocorticotropin from the pituitary By

activa-tion of CRF Receptor 1 (CRFR1) [2], CRF regulates not

only the endocrine, but also the autonomic, behavioural and

immune responses to stress [3] Moreover, accumulating

evidence indicates that CRF and its receptors play a

prominent role in the aetiology of several stress-related

disorders, such as depression and anxiety [4] CRFR1

belongs to family II of the G-protein coupled receptors

(GPCRs) This family is composed of several distinct peptide

receptors, such as secretin, parathyroid hormone and VPAC

(vasoactive intestinal polypeptide and pituitary adenylate

cyclase activating polypeptide receptors) Despite the impact

of CRFR1 on the stress-response and stress-related

dis-orders, little is known about its regulation In general, the

sensitivity of GPCRs to extracellular stimuli depends upon

various regulatory mechanisms including receptor desensi-tization, internalization and resensitization Although recent studies on CRFR1 desensitization revealed the involvement

of G-protein coupled receptor kinase 3 (GRK3) [5] and protein kinase C (PKC) [6], details of CRFR1 internalization remains uncharacterized

Internalization following agonist activation is a common phenomenon among GPCRs The process serves as the initial step of either receptor resensitization [7] or down-regulation

by lysosomal degradation Furthermore, reports have been made about internalization-induced signal transduction [8] According to studies mainly on the b-adrenergic receptors, belonging to the family I GPCRs, the process of agonist-induced GPCR internalization is facilitated by the same proteins as those involved in receptor desensitization Following activation, receptors are phosphorylated by specific GRKs at serine/threonine residues in the third intracellular loop and/or C-terminal tail GRK-mediated receptor phosphorylation promotes the binding of cytosolic b-arrestins, which not only uncouple receptors from their cognate G-proteins, but also target receptors for internal-ization through the subsequent interaction between the receptor/b-arrestin complex and proteins of the endocytic machinery such as clathrin [9] and the clathrin adaptor protein AP-2 [10] Though these features can be applied to several GPCRs [11], notable exceptions exist and mechanisms involved in the process of agonist-induced GPCR internal-ization is still a subject of controversy For example, whereas the b2-adrenergic receptor is internalized via clathrin coated pits [12], the cholecystokinin receptor [13], the bradykinin receptor [14] and the b-adrenergic receptors [15] have been

Correspondence to S M Nielsen, Department of Molecular

Pharma-cology, H Lundbeck A/S, 9 Ottiliavej, DK-2500 Valby, Denmark.

Fax: +45 3643 8253, Tel.: +45 3643 2096,

E-mail: smn@lundbeck.com

Abbreviations: b-arr1, b-arrestin 1; CRF, corticotropin releasing

factor; CRFR1, corticotropin releasing factor receptor 1; EGFP,

enhanced green fluorescence protein; GPCR, G-protein coupled

receptor; GRK, G-protein coupled receptor kinases; HEK, human

embryonic kidney; PKA, cAMP-dependent protein kinase;

PKC, protein kinase C.

(Received 9 July 2004, revised 31 August 2004,

accepted 20 September 2004)

demonstrated to internalize, at least in part, by the

caveolae-endocytic pathway Likewise, studies on the secretin receptor

indicate that GRK phosphorylation is not sufficient to

facilitate or mediate receptor internalization [16], suggesting

that kinases other than GRKs may play a greater role in

GPCR endocytosis than previously appreciated These

suggestions are supported by the recent finding that not only

GRK, but also cAMP-dependent protein kinase (PKA) are

involved in agonist-induced internalization of b1AR [15]

In the present study, we seek to reveal molecular and

cellu-lar mechanisms responsible for agonist-induced

internali-zation of CRFR1 By use of confocal microscopy cellular

distribution of CRFR1 and b-arrestin 1 are explored

Radio-ligand binding is applied to quantify receptor internalization

and the effect of second-messenger kinases are explored using

the PKA inhibitor H-89 and the broad spectrum kinase

inhibitor staurosporine Finally, a series of receptors with

mutations in the third intracellular loop and C-terminal tail

are constructed and examined for their ability to internalize

and recruit b-arrestin 1 Interestingly, we find that

agonist-induced internalization of CRFR1 does not depend on

puta-tive phosphorylation sites in the third intracellular loop and

C-terminal tail Moreover, our results indicate that

recruit-ment of b-arrestin 1 to the membrane following receptor

activation is not a prerequisite for CRFR1 internalization

Experimental procedures

cDNA constructs and mutagenesis

The coding sequence for CRFR1 was inserted into the

mammalian expression vector pCI (Promega) between

EcoRI and XbaI restriction sites The sequence encoding

the EQKLISEEDL peptide from c-myc was inserted

between residue 31 and 32 of the N-terminus The insertion

of the c-myc epitope at this position in the N-terminal region

of mouse CRFR1 has previously been demonstrated not to

alter the binding or signalling properties [17] An enhanced

green fluorescence protein (EGFP)-conjugated CRFR1

construct (CRFR1–EGFP) was created deleting the stop

codon of CRFR1 and situating EGFP in frame directly after

CRFR1 The fusion of EGFP to the C-terminus of numerous

other GPCRs reportedly does not alter receptor functionality

[18,19] Bovine b-arrestin 1 was a kind gift from T Schwartz

(University of Copenhagen, Denmark) An EGFP conjugate

(b-arr1–EGFP) was constructed by overlap PCR and the

product inserted into pCI-neo (Promega)

All mutant receptors were generated by PCR following

standard procedures [20] CRFR1-stop384 (C-terminally

truncated receptor after residue 384) was created using 3¢

antisense primers introducing a stop codon at the relevant

position followed by an XbaI restriction site CRFR1-IC3

was created by site-directed mutagenesis replacing the Ser,

Thr, Thr, Ser, Glu, Thr residues at positions 301–306 with

alanine residues Likewise, substitution of serine at position

372 for alanine was achieved by site-directed mutagenesis

All sequences were confirmed by automatic sequencing

Cell culture and transfection

Human embryonic kidney (HEK)293 cells (ATCC) were

grown in Dulbecco’s modified Eagle’s medium (DMEM)

with Glutamax I supplemented with 10% (v/v) heat inac-tivated fetal bovine serum, 5 mM sodium pyruvate and penicillin/streptomycin (100 lgÆmL)1) at 37C in a humidi-fied incubator with 5% CO2 All products for cell culture were from Gibco

For transfection, cells were plated in 90-mm plastic dishes at a density of 3.0· 106 cells per dish in medium without antibiotics for 18–24 h before use Transient transfection was carried out using the LipofectAMINE

2000 (Invitrogen Life Technologies, Carlsbad, CA) method according to the manufacturer’s instructions For studies visualizing receptor localization, 4 lg plasmid containing cDNA encoding CRFR1–EGFP was used In coexpres-sion studies, 2 lg b-arr1–EGFP was used in addition to

4 lg of the relevant CRFR1 construct Twenty micro-grams of all cDNA constructs were used for radioligand binding and functional assays Transfected cells were cultured for 48 h to allow expression Twenty-four hours before experiments cells were seeded in appropriate dishes or plates precoated with 20 lgÆmL)1 polyD-lysine (Sigma)

Drug treatment For studies including H-89, N-[2-((p-Bromocinnamyl)ami-no)ethyl]-5-isoquinolinesulfonamide, 2HCl (Calbiochem, VWR Denmark) and staurosporine (Sigma) drugs were added to the culture medium 20 min in advance in a concentration of 5 lMand 100 nM, respectively, and these concentrations were maintained throughout the experiment Human/rat CRF (BACHEM, Germany) was used at a final concentration of 10)8M

Immunocytochemistry For colocalization studies of the CRF receptor with b-arrestin 1, cells expressing c-myc epitope-tagged CRFR1 and b-arr1–EGFP were grown on poly D-lysine coated glass coverslips (Menzel-glaser) in 35-mm dishes at a density of 5.0· 105 cells per dish After incubation with

10)8MCRF for various times, cells were fixed in 4% (v/v) paraformaldehyde for 10 min, washed twice in NaCl/Pi and permeabilized with 0.1% (v/v) Triton X-100 in blocking buffer (1% BSA/NaCl/Pi) Subsequently, cells were incubated for 45 min with monoclonal mouse c-myc antibody (clone 9E10, Sigma) diluted 1 : 1000 (5.3 lgÆmL)1) in blocking buffer Following five washes

in blocking buffer, Cy3 (Indocarbocyanine)-conjugated antibody against mouse (Jackson) 1 : 200 was applied for another 45 min before the cells were washed twice in blocking buffer, twice in NaCl/Pi and mounted using Vectashield (Vector Laboratories, Burlingame, CA) Coverslips were sealed with nail polish Fluorescence was detected using confocal microscopy as described below Unspecific binding was tested by excluding either the primary or secondary antibody

Detection of EGFP in living cells

To visualize CRFR1–EGFP and b-arr1–EGFP in living cells, HEK293 transiently transfected with the relevant cDNA were plated on glass-bottomed culture dishes

(0.17-mm Delta T dish, Bioptechs Inc., Butler, PA) and kept

at 35C on a heated microscope stage during the

experi-ment CRF was added to the culture dish and images were

collected at the indicated time points using confocal

microscopy

Confocal microscopy

Confocal microscopy was performed on a Biorad

Radi-ance2000 confocal laser scanning microscope (CLSM) using

a Nikon 60· NA 1.4 oil immersion objective to examine

immuno-stained coverslips or a Nikon 60· NA1.2 water

immersion objective to examine living cells

EGFP was excited with 488-nm Ar laser and the

fluorescent signal was collected with an emission filter set

comprising a 560-nm long-pass dichroic mirror and a 500–

530-nm barrier filter For Cy3 detection the 543-nm Green

HeNe laser was used along with a 555–625-nm emission

filter In addition to the magnification provided by the

objectives an additional zoom factor of 1.8–3.0 was applied

Images were collected in 512· 512 pixels with a scan speed

of 50 Hz Pinhole was set to achieve the optimal confocal

sectioning, which is determined by the Airy disk diameter

However, sometimes the pinhole was opened in order to

improve light collection of preparations with a weak

fluorescence To adjust detector gain and offset, a

false-colour look-up table was applied To improve the

signal-to-noise ratio each image was an average of three or four

scans Images were subsequently processed using Adobe

PHOTOSHOP5.0 Lineprofile was performed using the

LASERPIXsoftware (Bio-Rad)

125

I-labelled sauvagine internalization

The day prior to the experiment transfected cells were plated

at a density of 200 000 cellsÆwell)1in 24-well dishes (Nunc

A/S Denmark) One hour prior to the assay, medium was

changed to assay medium (DMEM supplemented with

20 mM HEPES and 0.1% BSA) Internalization was

initiated by incubating with radiolabelled CRFR agonist

125I-labelled sauvagine (PerkinElmer) diluted to 100 000 c.p.m in 0.5 mL assay buffer for various times at 37C Subsequently, cells were transferred to ice and washed twice

in ice-cold NaCl/Pi To remove surface-bound radioligand, cells were washed with 1 mL of acid solution (50 mMacetic acid, pH 3) for 10 min The acid supernatant, containing surface-bound radioactivity, was collected and measured Subsequently, cells were solubilized in lysis buffer (0.2M NaOH, 2% NP40) and internalized radioactivity was measured Nonspecific binding, determined in the presence

of 10)7Munlabelled CRF, was subtracted and the radio-activity internalized was expressed as a percentage of the sum of the surface radioactivity and the internalized radioactivity In experiments where the effect of hypertonic medium was tested, cells were pretreated with 0.4Msucrose for 20 min and this concentration was maintained during radioligand incubation Data were analysed using Graph-PadPRISMsoftware

Results Visualization of agonist-mediated internalization

of CRFR1 The C-terminally EGFP-conjugated version of the receptor, CRFR1–EGFP, was used in order to visualize the cellular localization and trafficking of CRFR1 following agonist exposure Fluorescence was detected in transiently trans-fected HEK293 cells by confocal microscopy In unstimu-lated cells, the fluorescence was almost exclusively confined

to and sharply outlining the contours of the plasma membrane, as shown in Fig 1A1 Following CRF expo-sure, a time-dependent increase in the appearance of small fluorescent intracellular vesicles was observed (Fig 1A2 and A3) After 20 min of incubation, an aggregate of fluores-cence started to appear near the nucleus of each cell (A4) and after 40 min, these aggregates became even more distinct (A5) However, fluorescence was still detectable at

Fig 1 Agonist-induced redistribution of CRFR1–EGFP visualized by confocal microscopy HEK293 cells transiently expressing CRFR1-EGFP were exposed to 10)8M CRF for 0 (A1), 5 (A2), 10 (A3), 20 (A4) and 40 min (A5) In unstimulated cells (A1), CRFR1–EGFP was evenly distributed and concentrated at the plasma membrane Following agonist exposure, a time-dependent increase in intracellular CRFR1–EGFP was observed (A2–5) As a control, HEK293 cells transiently expressing EGFP alone were exposed to the same conditions (B1–5) The images are representative of multiple independent studies Bar ¼ 10 lm.

the membrane, although in a more punctuate pattern than

in unstimulated cells As a control, HEK293 cells transiently

expressing EGFP showed a relatively even distribution of

fluorescence throughout the cell and this distribution

remained unaltered during CRF exposure (Fig 1B)

Interestingly, the morphology of the

CRFR1–EGFP-expressing cells changed during the experiment The

unstimulated cells appeared unaltered compared to

un-transfected cells, whereas following stimulation the cells

seemed to loose the attachment to the bottom of the dish

and shrink (Fig 1A1 vs A5) This change in morphology

was not observed for cells expressing only EGFP stimulated

with CRF (Fig 1B)

Quantification of agonist-mediated CRFR1 internalization

To quantify receptor internalization, we used the CRF

analogue125I-labelled sauvagine for internalization studies

The use of radioligand internalization to assay receptor

internalization is based on the assumption that receptor and

ligand are endocytosed together and that the intracellular

receptor–ligand complex can be determined by measuring

intracellular radioactive labelled ligand

Following 125I-labelled sauvagine stimulation, within

5 min more than 50% of the cell specific associated

radioligand was internalized (Fig 2) The internalization

reached a plateau after 10–20 min, with a maximal

radio-ligand internalization of 69% Consistent with the

translo-cation of CRFR1–EGFP observed by confocal microscopy,

these results demonstrate that CRFR1 is internalized

following agonist exposure and that internalization can

take place within minutes

Agonist-induced endocytosis of many GPCRs occurs

via clathrin-coated pits, a process that can be inhibited

by hypertonic sucrose [21] Thus, in order to determine

if CRFR1 was internalized via clathrin-coated pits,

125I-labelled sauvagine incubation was performed in a medium containing 0.4M sucrose Under these circum-stances, almost no internalization of radioligand was observed during the initial 10 min and the maximal extent

of internalization was reduced to 39% following 40 min of incubation (Fig 2) This result indicates the involvement of clathrin-coated pits in the agonist-induced internalization

of the CRFR1

PKA and PKC inhibitors do not abolish agonist-induced CRFR1 internalization

As CRFR1 activation is thought to signal through both the cAMP and the PLC pathway [22], the second messenger product of either or both of these pathways could

be involved in agonist-stimulated internalization of this receptor

The effect of PKA and PKC was investigated by use of the specific PKA inhibitor H-89 and the broad-spectrum protein kinase inhibitor stauroporine Radioligand inter-nalization assay was performed as described, including H-89

or staurosporine in the culture medium before and during

125I-labelled sauvagine incubation As shown in Fig 3, no significant alteration of the extent of receptor-mediated internalization of125I-labelled sauvagine could be observed

in the presence of any of these inhibitors These findings indicate that neither the activation of PKA nor the activation of PKC is necessary for agonist-induced CRFR1 internalization

Involvement of putative phosphorylation sites

in agonist-induced receptor internalization

To explore further the impact of various serine/threonine residues on receptor internalization, we constructed a series

of CRFR1 with mutations in intracellular loop 3 and the C-terminal tail cAMP measurements (data not shown) indicate that these mutations do not significantly alter CRF-induced cAMP production The various constructs are

Fig 2 Quantification of agonist-induced CRFR1 internalization.

Transiently transfected HEK293 cells were incubated with125I-labelled

sauvagine for various times at 37 C Cell surface-bound and cytosolic

radioligand was determined and the specific internalization was

determined as described in Experimental procedures To examine the

effect of hypertonic media, 0.4 M sucrose was added before and during

incubation (A) Internalization of CRFR1 in the absence (j) and in

the presence of sucrose (m) is shown (B) The proportion of125

I-labelled sauvagine internalized after 40 min was 69% (± 2%), but

only 39% (± 3%) in the presence of sucrose (Students t-test:

***P < 0.005) Data represent the mean (± SEM) of four separate

experiments performed in triplicate.

Fig 3 The effect of the PKA inhibitor H-89 and the broad spectrum kinase inhibitor staurosporine on radioligand internalization Transiently transfected HEK293 cells were incubated with125I-labelled sauvagine

at 37 C for 40 min without or in the presence of 5 l M H-89 or 100 n M

staurosporine The proportion of 125 I-labelled sauvagine internalized after 40 min was 69% (± 2%), 66% (± 1%) in the presence of H-89 and 66% (± 2%) in the presence of staurosporine The extent of receptor internalization without inhibitors was set to 100% Data represent the mean (± SEM) of four independent experiments per-formed in triplicate.

depicted schematically in Fig 4A As shown in Fig 4B,

truncation of the C-terminal tail at position 384

(CRFR1-stop384), where all but one of the putative phosphorylation

sites in the C-terminal tail was removed, did not cause a

reduction in radioligand-induced internalization Neither

did an additional mutation of the remaining serine in close

proximity to the expected seventh transmembrane domain

(CRFR1-stop384; S372A) Furthermore, a construct with

substitution of a cluster of serines and threonines to alanines

in the third intracellular loop (CRFR1-IC3) retained its

ability to internalize to the same extent as the wild-type

CRFR1 Likewise a combination of this modification and

CRFR1-stop384 (CRFR1-stop384; IC3) did not affect

internalization Taken together, these data indicate the

ability of CRFR1 to internalize in the absence of the

majority of putative phosphorylation sites in the C-terminal

tail and third intracellular loop

b-arrestin 1 is recruited to the plasma membrane

following CRFR1 activation

To examine the cellular trafficking of b-arrestin 1 following

receptor activation, an EGFP conjugate of b-arrestin 1

(b-arr1–EGFP) was used HEK293 cells were transiently

cotransfected with b-arr1–EGFP and CRFR1 and cellular

localization of b-arr1–EGFP was visualized by confocal

microscopy In unstimulated cells, b-arr1–EGFP

fluoresc-ence was evenly distributed throughout the cytoplasm with

no apparent enhanced plasma membrane localization

(Fig 5A1) However, within minutes following receptor

stimulation b-arr1–EGFP was rapidly redistributed to the

plasma membrane and after 5 min the cytosol was almost

depleted (Fig 5A3) No significant b-arr1–EGFP

translo-cation was observed in cells lacking overexpressed CRFR1

(Fig 5B) This demonstrates that b-arr1–EGFP is recruited

specifically to the plasma membrane in response to CRFR1

activation

b-arr1–EGFP was never observed associated with intracellular vesicles following receptor activation This was readily demonstrated in a colocalization study of b-arr1–EGFP with c-myc-tagged CRFR1 (Fig 6) In unstimulated cells, CRFR1 immunofluoresence (red) was confined to the plasma membrane, whereas the b-arr1– EGFP fluorescence (green) was distributed in the cytoplasm (Fig 6A1) This is illustrated in the overlay and quantified

in the line profile by the peaks of CRFR1–Cy3, where the line crosses the plasma membrane and the even distribution

of b-arr1–EGFP fluorescence throughout the cytosol (Fig 6A2) In response to agonist activation of the CRFR1, b-arr1–EGFP was translocated to the membrane where it colocalizes with CRFR1 as visualized by the appearance of yellow spots (Fig 6B1) The colocalization is also demon-strated in the line profile in that the intensity of CRFR1– Cy3 and b-arr1–EGFP fluorescence peaks at the same position (Fig 6B2) However, yellow spots and colocaliza-tion of fluorescence intensity in the line profile could only be observed at or near the cell membrane No colocalization of b-arr1–EGFP with CRFR1 was observed in the CRFR1-containing intracellular vesicles in the cytoplasm of the cell (Fig 6B1) This is also illustrated in the line profile, where the increase in intracellular CRFR1–Cy3 fluorescence representing internalized CRFR1 is not colocalized with b-arr1–EGFP (Fig 6B2)

Involvement of putative phosphorylation sites

in recruitment of b-arrestin1

To examine if removal of potential phosphorylation sites

in the C-terminal tail and the third intracellular loop had an effect on recruitment of b-arrestin 1 following receptor acti-vation, trafficking of b-arr1–EGFP in response to CRF was visualized by live confocal microscopy in HEK293 cells coex-pressing b-arr1–EGFP and CRFR1-stop384:IC3 Fig 5C1 demonstrates that in unstimulated cells fluorescence is

Fig 4 Putative phosphorylation sites in C-terminal tail and third intracellular loop are not necessary for agonist-induced receptor internalization (A) Schematic representation of CRFR1 Mutated residues are shown in grey circles (B) HEK293 cells transiently expressing the wild-type or mutant CRFR1 were incubated with I125-labeled sauvagine for 40 min at 37 C Internalization of radioligand with the wild-type receptor was set to 100% Each bar represents the mean ± SEM of four independent experiments performed in triplicate (***, P < 0.005 Student’s t-test).

evenly distributed in the cytosol corresponding to the

distribution of b-arr1–EGFP coexpressed with the wild-type

CRFR1 (Fig 5A1) When exposed to CRF, only few

clusters appear at the cell surface of some cells No profound translocation of cytosolic b-arr1–EGFP, as observed when coexpressed with the wild-type CRFR1 (Fig 5A3), is

A1

B1

0 50 100 150

Distance (µm) A2

0 50 100 150

Distance (µm)

B2

25

Fig 6 b-arrestin1 does not colocalize with CRFR1 in endocytic vesicles HEK293 cells transiently coexpressing c-myc tagged CRFR1 and b-arr1-GFP were incubated with or without CRF for 10 min and fixed with paraformaldehyde b-arr1–Eb-arr1-GFP was visualized by its intrinsic fluorescence (green) and CRFR1 was visualized by immunocytochemistry with 9E10 anti-c-myc primary and Cy3-conjugated secondary antibodies (red) CRFR1/b-arr1–EGFP colocalization appears as yellow in the overlap image In unstimulated cells (A1), CRFR1 is confined to the plasma membrane, whereas b-arr1–EGFP is diffusely distributed in the cytosol After exposure to CRF for 10 min (B1), accumulation of CRFR1-containing intracellular vesicles is observed b-arr1–EGFP is translocated to the plasma membrane where it colocalizes with the remaining CRFR1, but no colocalization of b-arr1–EGFP and intracellular CRFR1 is observed The images are representative of three independent experiments Bar ¼ 10 lm Profiles of the fluorescence intensity along the lines depicted in the overlap images are shown (A2, B2).

Fig 5 Live confocal microscopy of HEK293

cells coexpressing b-arr1–EGFP and either

CRFR1 or CRFR1-stop384; IC3 In

unstimu-lated cells, b-arr1–EGFP cotransfected with

CRFR1 was evenly distributed in the

cyto-plasm (A1) Upon receptor stimulation,

b-arr1–EGFP was rapidly (within minutes)

redistributed to the plasma membrane (A2)

and after 5 min this redistribution was almost

complete (A3) As a control, the same

experi-ment was performed on cells expressing

b-arr1–EGFP alone (B1–3) No redistribution

upon CRF stimulation was observed in cells

not coexpressing CRFR1 When coexpressed

with the modified receptor, CRFR1-stop384;

IC3, b-arr1–EGFP was likewise distributed

evenly in the cytoplasm in unstimulated cells

(C1) However, shortly after stimulation

(2 min, C2) not much effect was observed and

after 5 min only a very small fraction of

b-arr1–EGFP was observed in clusters at the

plasma membrane of some cells (C3, arrows).

No further translocation was observed at later

time points The images are representative of

three independent experiments Bar ¼ 10 lm.

detectable (Fig 5C2,3) These observations indicate that

CRFR1-stop384; IC3 has a very limited capability of

recruiting b-arr1–EGFP to the membrane

Discussion

To date, most GPCRs characterized internalize following

agonist exposure However, despite the growing interest of

CRF and CRFR1 as essential components in the aetiology

of depression and other stress-related disorders,

internal-ization of CRFR1 has so far not been demonstrated In the

present study, we show agonist-mediated internalization of

CRFR1 Furthermore, we demonstrate internalization to be

independent of the second messenger-dependent protein

kinases PKA and PKC The extent of internalization

remained unaltered in mutated CRFR1 lacking putative

phosphorylation sites in the C-terminal tail and third

intracellular loop as compared to the wild-type receptor

However these mutations abolished the profound

recruit-ment of b-arrestin 1 to the plasma membrane observed in

response to stimulation of the wild-type receptor, indicating

that receptor phosphorylation and recruitment of b-arrestin

might be part of an event separate from that of CRFR1

internalization

Agonist-mediated internalization of CRFR1 was

dem-onstrated by visualizing CRFR1–EGFP conjugated

recep-tor and by quantification of radioligand internalization As

determined by use of radioligand, the extent of receptor

internalization reached almost its maximum after 5–10 min

of agonist exposure (Fig 2) Likewise, in response to

agonist stimulation, the CRFR1–EGFP fusion protein

moved from a homogenous plasma membrane distribution

to a punctuate distribution and location in small

intra-cellular vesicles This redistribution was detected after

5–10 min as visualized by confocal microscopy (Fig 1)

After 20–40 min, many of the small vesicles seemed to

fuse into larger structures observed near the nucleus

(Fig 1) The nature of these fluorescent aggregates is

unknown, but a similar pattern of fluorescence

distribu-tion was observed when EGFP-conjugated

thyrotropin-releasing hormone receptor was stimulated with agonist for

15–30 min [23] EGFP has a long half-life and the formation

of large fluorescence aggregates in the perinuclear region

could represent accumulation of EGFP-tagged CRFR1 in

lysosomes [24]

Interestingly, a profound change in cell morphology is

observed following stimulation with agonist (Fig 1) The

change is characterized by shrinkage of the cells and

detachment from the surface of the culture dish A similar

change in the shape of the cell following agonist stimulation

is observed with the Substance P receptor [25] Detachment

of the cells from the surface of the culture dish could reflect

disassembly of the actin cytoskeleton in the membrane

regions as a consequence of excessive membrane receptor

activation

Internalization of membrane proteins may occur by at

least two different mechanisms: receptor endocytosis via

clathrin-coated pits; or caveolae-mediated internalization

The clathrin-coated pits pathway is the best characterized

endocytic route and is used by numerous GPCRs [11] The

formation of the clathrin lattice is disrupted by hypertonic

sucrose and this is an established method used to block

internalization processes that involves clathrin-coated pits and vesicles [21] In the present study, sucrose in the media inhibited CRFR1 internalization by 50% indicating (Fig 2) that a part of CRFR1 internalization may be clathrin dependent Similar partial inhibition of internalization is observed for the parathyroid hormone receptor after treatment with sucrose [26] However, it is not clear if the remaining 50% reflects an alternative mechanism of inter-nalization, such as internalization via caveolae as seen for

b1-adrenergic receptor [15] and cholecystekinin receptor [13], or an incomplete inhibition of clathrin-coated pit formation Depletion of cholesterol levels prior to experi-ments could reveal the involvement of caveolae

Activation of many GPCRs, including CRFR1, leads to signalling via the cAMP and/or PLC pathways, which involve activation of the second messenger-dependent protein kinases PKA and PKC However, even though internalization of some family II GPCRs are dependent on PKA [16] and PKC [27], not much attention has been drawn

to the involvement of these kinases in GPCR internaliza-tion CRFR1 possesses several putative PKA/PKC sites in the third intracellular loop and C-terminal tail PKA preferentially phosphorylates serine and threonine residues with the basic residues arginine or lysine at position)2, )3 [28] and four serine residues in this consensus sequence are present in the third intracellular loop and in the C-terminal tail (Ser301 Ser396, Ser405 and Ser412) Two potential PKC phosphorylation sites (Ser386 and Ser408) are present in the C-terminal tail of CRFR1 [6] In our study, truncation of the C-terminal tail at position 384 and substitution of serine/ threonine residues for alanine in the third intracellular loop removed all putative PKA/PKC phosphorylation sites However, this did not alter the extent of receptor internal-ization (Fig 4) In addition, the PKA inhibitor H-89 and the broad spectrum kinase inhibitor staurosporine had no effect on the extent of agonist-induced CRFR1 internal-ization (Fig 3) Thus, internalinternal-ization of the family II GPCR CRFR1 does not seem to depend upon the second messenger kinases PKA and PKC

Recruitment of b-arrestin to the plasma membrane following receptor activation has been demonstrated for several GPCRs [29] The function of b-arrestin is to uncouple the receptor from the G-protein, thereby mediating receptor desensitization, and furthermore to couple the receptor to the endocytic machinery and thus mediate internalization In this study, activation of CRFR1 led to the recruitment of b-arrestin 1 to the plasma membrane also (Fig 5A) The initial redistribution of b-arrestin 1 was observed already after 2 min and no further changes in translocation was observed after minutes of agonist stimulation With the rate

of CRFR1 internalization reaching maximum after 5–10 min, this result is in agreement with recruitment of b-arrestin 1 preceding receptor internalization

For many GPCRs, b-arrestin is subsequently transferred along with receptor-containing endocytic vesicles [30] Although we demonstrated both CRFR1–EGFP and myc-tagged CRFR1 to redistribute from a diffuse mem-brane localization to intracellular vesicles, b-arr1–EGFP was never observed in association with intracellular vesicles following receptor activation (Fig 6) These findings indi-cate that b-arrestin 1 is recruited to the receptors at the plasma membrane following receptor activation, but that

the CRFR1/b-arrestin 1 complex dissociates at or near the

plasma membrane before reaching the endocytic vesicles

This phenomenon has been observed for other GPCRs,

such as the b2-adrenergic receptor [30] The difference in

stability of the b-arrestin/receptor complex for different

GPCRs appears to depend on a cluster of serine/threonine

residues in the C-terminal tail [31] Because such a cluster is

not present in the C-terminal tail of CRFR1, it might

explain why stable b-arrestin/receptor complex is not

detected in our experiments The exact mechanisms

respon-sible for the dissociation of b-arrestin and receptor are

currently unknown, but the biological effect seems to be

rapid receptor dephosphorylation and recycling [32]

Binding of visual arrestin to rhodopsin requires GRK

phosphorylation of rhodopsin Several other studies have

confirmed the role of GRK phosphorylation in binding of

b-arrestin to GPCRs [33] The substrate of GRK

phos-phorylation is usually serine and threonine residues within

the C-terminal tail and/or the third intracellular loop In

accordance with this theory, cells coexpressing

CRFR1-stop384:IC3 and b-arrestin1 showed marked impairment of

b-arrestin1 recruitment to the membrane following receptor

stimulation (Fig 5C) However, the recruitment was not

completely abolished Some clusters of b-arrestin 1 could

still be observed at the membrane in response to receptor

activation even in the absence of the C-terminal tail of the

receptor and the cluster of serine/threonine in the third

intracellular loop This could be due to heterologous

recruitment to other endogenously expressed receptors in

response to CRFR1 activation However, the possibility

remains that CRFR1 contains determinants of b-arrestin 1

interaction apart from the C-terminal tail and/or serine/

threonine in the third intracellular loop that are sufficient

for transient association Nevertheless, regions in the

C-terminal tail/third intracellular loop are required for a

more profound translocation of b-arrestin1 to the receptor

Interestingly, removal of putative phosphorylation sites in

the C-terminal tail and/or IC3 did not alter the extent of

receptor internalization (Fig 4) This is in contrast to the

prevailing observations of GPCRs dictating both the process

of desensitization and of internalization to depend on

receptor phosphorylation [34,35] and recruitment of

b-arrestin [36] In accordance with our studies a report on

the parathyroid hormone receptor indicates that a lysine in

the third intracellular loop and an asparagine in the third

transmembrane helix are important for receptor

internal-ization [37] Both residues are conserved among family II

GPCRs, including the human CRFR1 In the VPAC1

receptor, a serine in the C-terminal tail seems to be important

for receptor desensitization, but mutation of this serine does

not affect agonist-mediated VPAC1 receptor internalization

This observation along with our data suggest that, at least

for some family II GPCRs, desensitization and

internalizat-ion might be mediated by two separate mechanisms

Phosphorylation and recruitment of b-arrestin serve to

uncouple the receptor from the G-protein, whereas other yet

unidentified mechanisms serve to render the receptors to

endocytic vesicles, thus enabling receptor regulation by

degradation or recycling of endocytosed receptors

In summary, internalization of CRFR1 in HEK293 cells

in response to agonist has been demonstrated The process

seems to involve clathrin-coated pits, but to be independent

of the activation of PKA and/or PKC b-arrestin 1 is recruited to the receptors following activation, but no colocalization of b-arrestin 1 with receptor-containing endocytic vesicles could be observed Whereas profound recruitment of b-arrestin 1 following CRFR1 activation was dependent on a cluster of serine/threonine residues in the third intracellular loop and/or residues in the C-terminal tail, agonist-induced CRFR1 internalization was not, sug-gesting that CRFR1 internalizes independently of receptor phosphorylation and interaction with b-arrestin 1 Acknowledgements

We thank Jacob Flemming Hansen for superior help with figure preparations and Kate Christensen and Louise Tarpø for excellent technical assistance.

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