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Wolfson Centre for Age-Related Diseases, King's College London, Guy's Campus, London Bridge, London, United Kingdom
Abstract
We have studied truncation mutants of the rat neuropeptide Y (NPY) Y1 receptor lacking four (Thr361stop, Y1T361) or eight (Ser352stop, Y1S352) potential serine/threonine C-terminal phosphorylation sites. NPY-stimulated hemagglutinin-tagged Y1, Y1T361, and Y1S352 receptors all efficiently activated G proteins in Chinese hamster ovary (CHO) cell membranes, but desensitization after NPY pretreatment was only prevented in the HAY1S352 clone. In transfected colonic carcinoma epithelial layers, functional Y1 and Y1T361 peptide YY responses became more transient as the agonist concentration increased, whereas those mediated by the Y1S352 receptor remained sustained. NPY-stimulated HAY1 receptor phosphorylation was increased by transient overexpression of G protein-coupled receptor kinase 2, and only Ser352stop truncation abolished this response in CHO or human embryonic kidney (HEK) 293 cells. Rapid internalization of cell-surface HAY1 receptors in HEK293 cells was observed in response to agonist, resulting in partial colocalization with transferrin, a marker for clathrin-mediated endocytosis and recycling. It is surprising that both truncated receptors were constitutively internalized, predominantly in transferrin-positive compartments. NPY increased cell-surface localization of HAY1S352 receptors, whereas the distribution of both mutants was unaltered by BIBO3304. Recruitment of green fluorescent protein-tagged -arrestin2 to punctate endosomes was observed only for HAY1 and HAY1T361 receptors and solely under NPY-stimulated conditions. Thus, the key C-terminal sequence between Ser352 and Lys360 is a major site for Y1 receptor phosphorylation, is critical for its desensitization, and contributes to the association between the receptor and -arrestin proteins. However, additional -arrestineCindependent mechanisms control Y1 receptor trafficking under basal conditions.
Neuropeptide Y (NPY)-related peptides comprise both a neuromodulator (NPY) and hormones [peptide YY (PYY) and pancreatic polypeptide] whose widespread distribution contributes to a diversity of physiological functions. They act in humans through four cloned Y-receptor subtypes (Y1, Y2, Y4, and Y5), members of the class A seven transmembrane domain (7TM) receptor family that signal through pertussis toxin-sensitive G proteins (Michel et al., 1998). Of these, the Y1 receptor was the first to be cloned (Krause et al., 1992) and remains the subtype for which the greatest range of selective agonists (Michel et al., 1998), antagonists (e.g., BIBO3304) (Wieland et al., 1998b), and knockout models (Pedrazzini et al., 1998) are available. These multiple approaches have defined important roles for central Y1 receptors in the feeding response to NPY released from hypothalamic arcuate neurones (Pedrazzini et al., 1998; Wieland et al. 1998b), and NPY-induced anxiolysis (Gibbs et al., 2004). In the periphery, this subtype contributes to sympathetic vasoconstriction (Pedrazzini et al., 1998) and to the inhibition of gastrointestinal secretion (Cox and Tough, 2002) by both neuronal NPY and PYY released from colonic endocrine cells. Y1 receptor activation also has long-term consequences, mediating the neuroproliferation of olfactory progenitors (Hansel et al., 2001) and regulating the growth of intestinal epithelial cells (Mannon, 2002).
Although Y receptors activate a very similar repertoire of Gi/o-linked second-messenger and ion-channel responses, the regulatory mechanisms defining the extent of this signaling may differ substantially between the subtypes (Michel et al., 1998). For example, Y1 receptor responses rapidly wane after prolonged agonist exposure whether expressed endogenously (Michel, 1994) or transfected (Gicquiaux et al., 2002; Holliday and Cox, 2003), a process known as desensitization. The Y1 receptor is also readily sequestered inside the cell when occupied by different ligands (Parker et al., 2001; Gicquiaux et al., 2002; Pheng et al., 2003). In contrast, the Y2 receptor, which also binds NPY and PYY with high affinity, undergoes relatively less desensitization and endocytosis (Parker et al., 2001; Gicquiaux et al., 2002). Berglund et al. (2003) provided one explanation for these differences by demonstrating that agonist-occupied Y1 receptors interact with the protein -arrestin2 much more efficiently than the Y2 subtype. By preventing contact between the stimulated receptor and G protein, the recruitment of -arrestin family members mediates the desensitization of many 7TM receptors (Shenoy and Lefkowitz, 2003; Gurevich and Gurevich, 2004). -Arrestins also guide receptor internalization via coated pits (through additional binding sites for phosphoinositides, clathrin, and accessory proteins such as AP-2), and they participate as molecular scaffolds for protein kinase cascades signaling to the cytoplasm and nucleus (Shenoy and Lefkowitz, 2003). They are the best known of an expanding array of partners that bind 7TM receptors directly, other than heterotrimeric G proteins (Brzostowski and Kimmel, 2001). Understanding how different 7TM receptors regulate these additional interactions is key to defining the full array of signaling pathways available to them and how the nature of their trafficking influences responses over time.
Since the pioneering studies on the 2-adrenoceptor (Bouvier et al., 1988), the C terminal region has emerged as a crucial domain governing the binding of many 7TM receptors to -arrestins (Braun et al., 2003; Kisselev et al., 2004; Neuschafer-Rube et al., 2004) and other regulatory proteins (Xiang et al., 2002). However, little is known about the molecular determinants that dictate the desensitization and trafficking of different Y-receptor subtypes. Here, we address the regulatory role of the Y1 receptor C-terminal domain by comparing the full-length receptor with two truncated mutants. We identify a key phosphorylated motif that is essential for its desensitization in biochemical and functional assays and for -arrestin2 binding. Our findings also reveal the regulation of constitutive Y1 receptor internalization by an arrestin-independent mechanism.
Materials and Methods
Materials. cDNAs for the rat Y1 receptor and -arrestin2 tagged with green fluorescent protein (GFP) at the N terminus were kindly provided by Professor T. Schwartz (Laboratory for Molecular Pharmacology, Copenhagen, Denmark); G protein-coupled receptor kinase 2 (GRK2) and GRK2(K220R) cDNAs were gifts from Professor S. Nahorski (University of Leicester, Leicester, UK). Molecular biology reagents were purchased from New England Biolabs (Hitchin, UK), Roche Molecular Biochemicals (Lewes, UK), or QIAGEN (Crawley, UK), with the exception of vectors pCruz-HA (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and pGEM-T (Promega, Southampton, UK). The rat monoclonal antibody against the hemagglutinin (HA) epitope (clone 3f10) was obtained from Roche, and transferrin-Texas Red and fluorophore-conjugated secondary antibodies were from Molecular Probes Invitrogen (Paisley, UK). Cell-culture reagents were sourced as follows: Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Paisley, UK), DMEM/Ham's F-12 medium, G418 sulfate, and L-glutamine (Sigma-Aldrich, Poole, Dorset, UK); fetal bovine serum of Australian origin (Invitrogen); trypsin (Lorne Diagnostics, Reading, UK); and other antibiotics (MP Biomedicals, Oxford, UK). 125I-PYY (2200 Ci/mmol) and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPS, 1250 Ci/mmol) were supplied by PerkinElmer Life and Analytical Sciences (Boston, MA), and both were stored in single-use aqueous aliquots at eC20°C; H 323PO4 (10 mCi/ml) was from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Peptides, stored as frozen aliquots of aqueous solution, were from Bachem (Merseyside, UK); UK14,304 (Sigma-Aldrich) was prepared as a 10 mM stock solution in dimethyl sulfoxide. BIBO3304 and piretanide were gifts from Boehringer Ingelheim Pharma KG (Biberach, Germany) and Aventis (Strasbourg, France), respectively. Calphostin C was purchased from Calbiochem (Nottingham, UK), and other reagents were from Sigma-Aldrich or VWR International (Poole, UK).
Truncated Y1 Receptor Constructs. Site-directed mutagenesis was performed with the Transformer mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA), using the rat Y1 receptor cDNA cloned into pGEM-T as a template (Holliday and Cox, 2003) and the following primers (with mutated nucleotides shown in boldface type and diagnostic restriction sites underlined) to introduce stop codons in place of Ser352 (5'-GACTATAGCCATGTGAAGCTTGCATACGGACGTG-3', HindIII) and Thr361 (5'-CGGACGTGTCCAAGTAATATTTGAAGCAGGCAAGCCCG-3', SspI). The BstX1/KpnI fragment containing the substitutions was excised and ligated into mammalian expression vectors pTEJ8-rY1 (which places the native Y1 receptor cDNA under the control of the ubiquitin C promoter) and pCruz-HAY1, in which the rat Y1 receptor cDNA is modified to include an N-terminal HA epitope (YPYDYPDVA), as described previously (Holliday and Cox, 2003). These cDNA constructs were each verified by double-stranded sequencing.
Cell Culture. Cells were maintained in DMEM containing 10% fetal bovine serum (HEK293 and HCA-7 colony 1 adenocarcinoma cells, a gift of Dr. S. Kirkland, Imperial College London, UK) (Marsh et al., 1993) or DMEM/F-12 supplemented with L-glutamine (200 mM) and 10% heat-inactivated fetal bovine serum [Chinese hamster ovary (CHO) K1 cells, kindly provided by Professor S. Hill, University of Nottingham, UK] as described previously (Holliday and Cox, 2003). Transfections were performed by calcium phosphate coprecipitation and glycerol (15%) shock, followed by selection, as appropriate, in 0.8 mg/ml G418 sulfate for 7 to 10 days. Individual stable colony 1 clones (transfected with untagged Y1 receptors) were isolated directly and screened for Y1 receptor function in short-circuit current (ISC) measurements. Single CHO and HEK293 clones (HA-tagged receptor constructs) with similar levels of 125I-PYY binding were generated by limiting dilution.
125I-PYY and [35S]GTPS Binding. These assays are described in detail by Holliday and Cox (2003). In brief, competition 125I-PYY binding experiments were performed by incubating fresh membrane preparations (120 min, 21°C) in buffer, pH 7.4 containing 8 mM HEPES, 4 mM KCl, 20 mM NaHCO3, 1.0 mM MgSO4, 1.0 mM K3PO4, 2.0 mM CaCl2, 0.2% bovine serum albumin (BSA) (HEK293, CHO) or 0.4% (w/v) (colony 1), and bacitracin (0.1 mg/ml). Radioligand concentration was 10 pM (CHO) or 25 pM (colony 1), and bound 125I-PYY was separated by filtration. Saturation assays in duplicate (CHO, HEK293 clones) measured 125I-PYY binding at a range of concentrations (0.01 to 5 nM, diluting the specific activity 20-fold with unlabeled PYY), determining nonspecific binding in the presence of 1 e BIBO3304.
Measurements of [35S]GTPS binding to CHO membrane aliquots (15eC30 e蘥 of protein) were performed in triplicate. In desensitization experiments, cells were incubated with or without 100 nM NPY in DMEM for 10 min at 37°C and washed extensively (2x DMEM, 3x phosphate-buffered saline, PBS) before membrane preparation. The optimized assay was performed in 10 mM HEPES buffer, pH 7.4, containing 50 or 100 mM NaCl, 10 mM MgCl2, GDP (1 or 10 e), 0.1 mM EDTA, 0.2% BSA, and 0.1 mg/ml bacitracin. After preincubation (90 min, 21°C) with or without agonist and/or antagonist, [35S]GTPS (200 pM) was added for 20 min before termination of the reactions by filtration. Filters were dried before the addition of scintillant (Ultima Gold MV; PerkinElmer Life and Analytical Sciences), and after 24 h, the retained radioactivity was determined in a -counter.
Measurement of Anion Secretion in Epithelial Layers. Epithelial layers of colony 1-transfected clones grown on permeable filters (area, 0.2 cm2) were mounted in Ussing chambers, bathed in oxygenated (95% O2/5% CO2) Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, and 11.1 mM glucose, pH 7.4) at 37°C and voltage-clamped at 0 mV (WP Instruments, Longmont, CO) as described previously (Holliday and Cox, 2003). Cell responses to vasoactive intestinal polypeptide (VIP), PYY, and other agents were measured continuously as changes in ISC, equivalent to the net electrogenic ion transport across the epithelial barrier.
Detection of Phosphorylated Receptors. For phosphorylation analysis, CHO or HEK293 clones were seeded in six-well plates, and where appropriate, they were also transiently transfected with GRK2 or GRK2(K220R) cDNAs (10 e蘥/well) the next day, 48 h before the experiment. On reaching 80% confluence, cells were loaded with 50 e藽i H 323PO4 in phosphate-free Krebs' buffer for 1 h at 37°C. Calphostin C (0.5 e) was included in the buffer for the final 30 min of the loading period where necessary. Peptides and BIBO3304 were subsequently added for the times specified in the text. After two washes in ice-cold phosphate-buffered saline, cells were disrupted in radioimmunoprecipitation assay buffer [50 mM Tris, 100 mM NaCl, 50 mM NaF, 10 mM Na4P2O7, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 200 e activated Na3VO4, 100 nM okadaic acid, and 10 e蘥/ml leupeptin and aprotinin, pH 8.0] by repeated passage through a 21-gauge needle. The solubilized extracts were rotated end-over-end for 2 h at 4°C, clarified by centrifugation (20,000g, 15 min, 4°C), and equalized for protein content (BCA protein assay; Pierce, Cheshire, UK). Immunoprecipitations were carried out overnight at 4°C by the addition of directly conjugated anti-HA agarose (clone 3f10), and then the precipitates were washed (4°C, 15 min, rotating) twice with radioimmunoprecipitation assay buffer, twice with high salt-wash buffer (50 mM Tris, 500 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, and 0.05% deoxycholate, pH 8.0), and once with low salt-wash buffer (50 mM Tris, 5 mM EDTA, 0.1% Nonidet P-40, and 0.05% deoxycholate, pH 8.0). Samples containing equal numbers of receptors were denatured in Laemmli loading buffer (80°C, 3 min) and resolved by SDS-polyacrylamide gel electrophoresis (10% Tris-HCl Ready Gels; Bio-Rad, Hemel Hempstead, UK). The dried gels were exposed to preflashed Amersham Hyperfilm MP for 24 to 72 h at eC70°C to detect 32P-labeled proteins. Proteins immunoprecipitated under the same conditions were also probed on Western blots with Y1 antibody CT/2 (a gift of Professor A. Beck-Sickinger, University of Leipzig, Leipzig, Germany) (Wieland et al., 1998a) or anti-HA (both detected by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence) to ensure equal receptor loading. The relative density of bands on scanned Western blots or autoradiographs was analyzed by Scion Image (version 4.0; Scion Corporation, Frederick, MD).
Immunofluorescence Microscopy. HEK293 clones were first grown to 50 to 70% confluence on poly-L-lysineeCcoated glass coverslips. Coverslips were transferred to humidified 35-mm Petri dishes and treated for 1 h in serum-free DMEM at 37°C before live labeling with anti-HA antibody (8 e蘥/ml, 30 min at 37°C) in serum-free DMEM /1% BSA in the presence of concanavalin A (0.3 mg/ml) or BIBO3304 (1 e) as appropriate. Y1 receptor ligands (with or without transferrin-Texas Red, 1:250) were then added for 5 to 30 min at 37°C. Two washes with ice-cold PBS terminated trafficking, and cells were subsequently fixed (2% paraformaldehyde in PBS, 15 min; quenched by PBS/25 mM glycine; 2 x 5 min) and permeabilized (0.075% Triton X-100 in PBS, 5 min). The primary antibody was visualized with goat anti-rat IgG-Alexa 488 (1:200 in PBS/1% BSA; 30 min at 21°C). Cells were postfixed in 2% paraformaldehyde, nuclear DNA stained with 4',6-diamidino-2-phenylindole, and coverslips were mounted in Mowiol 40-88 (Calbiochem). For studies of -arrestin2 translocation, HEK293 clones were transiently transfected with GFP--arrestin2 cDNA (10 e蘥/25-cm2 flask) 24 h before splitting onto coverslips. After 48 h, cells were treated with 1 e NPY for 5 to 30 min at 37°C, fixed, and mounted as described above.
Immunofluorescent analysis was performed as described in detail previously (Sunyach et al., 2003). In brief, a vertical stack of 25 to 30 fluorescent images was acquired digitally on a Zeiss Axiovert 100 microscope (63x oil objective, Omega Optical excitation and emission filter sets; Carl Zeiss GmbH, Jena, Germany), using Openlab 2.0 (Improvision, Coventry, UK) to direct a piezo z-axis drive in 0.2-e steps. The central 15 images (or 10 for studies of GFP-tagged -arrestin2 localization) for each fluorophore were then deconvolved to remove out-of-focus light (Openlab) and reconstructed in three dimensions (3.0 e z-section) in Volocity (Improvision).
Analysis. Displacement 125I-PYY binding curves and concentration-response curves in [35S]GTPS binding studies were analyzed as combined data groups from individual triplicate experiments using the program GraphPad Prism version 3.0 (GraphPad Software Inc., San Diego, CA) to yield pIC50 or pEC50 values ± 1 S.E.M. Total 125I-PYY binding sites (Bmax) estimates were obtained from single-site saturation curves (CHO, HEK293 clones) or from PYY displacements in colony 1 clones, where Bmax = TSB x IC50/[L], TSB is the total specific binding in the absence of agonist, and [L] is the free radioligand concentration. Concentration-response curves in electrophysiological studies were constructed from responses to single additions of the desired agonist and fitted by nonlinear regression (sigmoidal dose-response of variable slope; GraphPad Prism). Unpaired Student's t tests (two data groups) or one-way analysis of variance followed by Bonferroni's post hoc test (multiple comparisons) assessed statistical significance.
Results
The Distal Y1 Receptor C Terminus Does Not Influence G Protein Signaling. We constructed two truncation mutants of the HA-tagged rat Y1 receptor, in which Ser352 (HAY1S) and Thr361 (HAY1T) were replaced by stop codons (Fig. 1). When stably expressed in CHO cells at 4 to 5 pmol/mg, HAY1, HAY1S, and HAY1T receptors exhibited similar affinities for PYY, NPY, and the Y1 antagonist BIBO3304 in 125I-PYY competition experiments (Table 1). 125I-PYY binding to both wild-type and mutant receptors was significantly disrupted by a maximum concentration of 1 e GTPS (45eC60%; Table 1), a measure of the extent to which high-affinity agonist binding is promoted by G proteins (Holliday and Cox, 2003). We also assessed Y1 receptor G protein coupling directly using the [35S]GTPS binding assay. Under buffer conditions (100 mM NaCl, 10 e GDP) that maximized agonist-induced activation (Fig. 2, AeCC), basal binding was 77.9 ± 7.3 fmol/mg (n = 6) in CHO-HAY1, 81.6 ± 10.6 fmol/mg (n = 7) in CHO-HAY1S, and 68.9 ± 5.1 fmol/mg (n = 9) in CHO-HAY1T membranes. NPY concentration-response curves were similar in potency for HAY1 (pEC50: 8.33 ± 0.07, n = 4), HAY1T (pEC50: 7.66 ± 0.06, n = 4), and HAY1S receptors (pEC50: 7.98 ± 0.05, n = 4), whereas in all cases, PYY exhibited lower efficacy than NPY, particularly in CHO-HAY1S membranes. BIBO3304 prevented 1 e NPY responses in each of the three clones (Fig. 2, AeCC) and also acted as a weak negative agonist. For example, in buffer containing 50 mM NaCl and 1 e GDP, BIBO3304 attenuated basal [35S]GTPS binding to a similar extent in membranes from CHO-HAY1 (pEC50: 8.21 ± 0.41; 100 nM maximum 12.1 ± 2.9%, n = 3) and CHO-HAY1S clones (pEC50: 9.04 ± 0.38; 100 nM maximum 9.0 ± 1.7%, n = 3).
pKd and Bmax values were calculated from the direct fit to saturation experiments performed with 0.05 to 5.0 nM 125I-PYY, in which nonspecific binding was assessed with 1 e BIBO3304. PYY, NPY, and BIBO3304 pIC50 values are given for competition assays performed with 10 pM radioligand; 1 e GTPS attenuated total 125I-PYY binding (expressed as percentage inhibition of total binding) without altering the pIC50 values for competing PYY significantly (range, 9.22-9.57). Data are expressed as mean ± S.E.M. from the number of experiments indicated in parentheses.
HAY1S Receptors Are Resistant to Desensitization. We also investigated [35S]GTPS responses in membranes from cells that had been subjected to a short desensitizing pretreatment with NPY (100 nM, 10 min at 37°C). Compared with controls that had received vehicle alone, pretreated CHO-HAY1 membranes exhibited a substantial reduction in the maximal 1 e NPY response (Fig. 2D), with only a small change in the pEC50 value (control 8.71 ± 0.05 versus pretreated 8.36 ± 0.11, n = 5) in buffer containing 50 mM NaCl and 1 e GDP. The absence of an increase in basal [35S]GTPS binding (control 216.4 ± 15.6 fmol/mg versus NPY-treated 227.4 ± 10.0 fmol/mg, n = 5) or an enhanced sensitivity of this binding to BIBO3304 (control 3.8 ± 2.8% inhibition versus pretreated 3.9 ± 3.7%, n = 5) confirmed that the first agonist addition had been adequately removed. The attenuated NPY responses in pre-exposed membranes were therefore a consequence of the loss of functionally coupled HAY1 receptors. Although an identical reduction in maximal NPY response was observed in pretreated CHO-HAY1T membranes, agonist stimulation mediated by the HAY1S receptor was much less affected (Fig. 2, E and F). Thus, these experiments implicated the C-terminal region between Ser352 and Thr361 as a critical sequence governing Y1 receptor desensitization.
Expression of Truncated Y1 Receptors in Epithelial Cells. Three stably transfected clones of the HCA-7 colony 1 cell line were generated that expressed the wild-type Y1 (C1Y1), Y1T361 (C1Y1T), and Y1S352 receptors (C1Y1S) at moderate levels (PYY pIC50: 8.16eC8.33 and Bmax of 340 ± 120 (C1Y1; n = 6), 255 (C1Y1T; n = 2) and 135 fmol/mg (C1Y1S; n = 2) from 125I-PYY competition experiments). As confluent epithelial layers, these cells secrete chloride ions after elevations in intracellular cAMP and Ca2+. Responses are followed in real time by ISC techniques, allowing Y-receptor function to be studied at a signaling endpoint (Holliday and Cox, 2003).
Basal parameters varied between clones and the parent colony 1 cell line (mean resistance range of 39.1eC66.5 · cm2, initial ISC of 6.5eC22.7 e藺/cm2; n = 72eC531), as we have observed previously for stably transfected subpopulations (Holliday and Cox, 2003). Each clone also responded to VIP, an agonist that elevates cAMP, with similar pEC50 values (8.12eC8.51; n = 3eC69). Peak responses to VIP (30 nM) were sensitive to the loop diuretic piretanide, indicating that much of the ISC response was generated by electrogenic CleC secretion (Table 2 and Fig. 3). In contrast to host colony 1 epithelial layers, in which functional PYY responses are absent (Table 2), basolateral addition of PYY inhibited VIP-stimulated ISC in C1Y1, C1Y1T, and C1Y1S clones (Fig. 3). PYY pEC50 values (derived from pooled single-addition concentration-response relationships) were 7.70 ± 0.07 (C1Y1; n = 5eC8), 7.53 ± 0.30 (C1Y1S; n = 3eC5), and 7.64 ± 0.33 (C1Y1T; n = 3eC8). The maximal responses to 100 nM PYY (Table 2) were significantly lower in both truncated Y1 receptor clones compared with C1Y1 epithelial layers, as were maximal ISC decreases to 1 e UK14,304 (an agonist acting at endogenous 2-adrenoceptors, pEC50 range of 6.95eC7.25 between clones; n = 3eC5) (Table 2). Thus, these differences reflected more general variation between the size of Gi-mediated antisecretory responses of individual clones rather than a specific effect of Y1 receptor truncation.
The peak changes in ISC in response to VIP, PYY, and the 2-adrenoceptor agonist UK14,304 are indicated for nontransfected colony 1 epithelial layers and the three clones expressing Y1, Y1(S352), and Y1(T361) receptors. Data are expressed as mean ± S.E.M., with n values indicated in parentheses.
A selective analog with high affinity for Y1 receptors, [Leu31, Pro34]PYY (100 nM) (Michel et al., 1998), also decreased VIP-stimulated ISC (e.g., in C1Y1 cells, eC11.3 ± 0.9 e藺/cm2, n = 3), whereas 10-min preincubation with BIBO3304 (300 nM) abolished subsequent 100 nM PYY responses in each clone (n = 3; data not shown). Significant responses to PYY (100 nM) were only obtained when peptide was added to the basolateral reservoir. The small apical responses observed (e.g., in C1Y1 cells, eC1.5 ± 0.3 e藺/cm2 and in C1Y1S, eC1.2 ± 0.2 e藺/cm2; each n = 3) did decrease the subsequent sensitivity to a basolateral application (e.g., basolateral 100 nM PYY 10 min after apical in C1Y1S was eC2.0 ± 0.6 e藺/cm2 (n = 3); P < 0.05 versus control, eC5.4 ± 0.9 e藺/cm2; n = 5), suggesting that they arose from the stimulation of basolateral receptors after leakage of peptide through the epithelial barrier.
S352 Truncation Generates a Sustained Functional Y1 receptor Phenotype. Increasing concentrations of PYY initiated markedly more transient reductions in VIP-stimulated ISC in C1Y1 cells (Fig. 4A). An identical change to a short-lived PYY time course was also apparent in the C1Y1T clone (Fig. 4B), but in contrast, responses mediated by the Y1(S352) receptor remained sustained at the highest PYY concentration investigated (300 nM) and over at least a 10-min period (Fig. 4C). When normalized as a percentage of the peak response, significant differences between C1Y1S and either C1Y1 or C1Y1T 100 nM PYY time profiles were evident within 4 min of agonist addition [after peak reductions at 2eC3 (C1Y1, C1Y1T) or 4 min (C1Y1S)] and were maintained thereafter (Fig. 4D). In contrast to the more sustained phenotype of transfected Y1(S352) receptors, the time courses of 1 e UK14,304 responses in the C1Y1S clone were essentially identical with those in C1Y1 and C1Y1T cells, decaying to approximately 50% of the peak response 10 min after agonist application (Fig. 4E).
HAY1 Receptor Phosphorylation Is Prevented by Ser352 Truncation. We next assessed the incorporation of 32Pi into HAY1 receptors immunoprecipitated with anti-HA from solubilized extracts of CHO-HAY1 cells (Table 1) or a stable HEK293 clone (HEK-HAY1; 125I-PYY Bmax = 1.5 ± 0.2 pmol/mg, n = 3). Both host cell types yielded similar results, and only those from HEK293 cells are illustrated.
No phosphorylated proteins were identified in immunoprecipitates from nontransfected HEK293 cells, and only a faint specific band of 71 to 100 kDa (n = 4) was observed for the HEK-HAY1 clone under basal conditions. This corresponded to the major mature receptor protein detected by Western blotting with either anti-HA or anti-CT/2 (a polyclonal antibody directed against the Y1 receptor C terminus) (Wieland et al., 1998a). When the cells were stimulated by 1 e NPY before solubilization and immunoprecipitation, the labeling intensity of this band was substantially increased (Fig. 5), a response which was rapid (maximal for 1-min agonist treatments) and maintained for longer incubation periods (up to 15 min; data not shown). HA-Y1 receptors were also phosphorylated after PYY addition (1 e response at 1 min: 2.0-fold over basal, compared with 3.6-fold for 1 e NPY in the same two experiments), and agonist-induced phosphorylation was prevented by 10-min preincubation with 1 e BIBO3304 (n = 2; data not shown).
We also observed agonist-dependent 32Pi-labeling of HAY1T receptors in the HEK-HAY1T clone (125I-PYY Bmax = 2.0 ± 0.2 pmol/mg; n = 3), with 1-min NPY treatment resulting in a predominant band of slightly lower molecular mass than in HEK-HAY1 cell extracts (70eC89 kDa; n = 3). However, under the same stimulated conditions, HAY1S receptors were not significantly phosphorylated, either in the HEK-HAY1S clone (125I-PYY Bmax = 1.2 ± 0.2 pmol/mg; n = 3) (Fig. 5A) or in CHO-HAY1S cells (n = 2; data not shown).
To investigate the kinases involved in phosphorylating the Y1 receptor, we transiently transfected cDNAs for GRK2 or its inactive mutant K220R into HEK-HAY1 cells. Overexpression of GRK2 substantially enhanced NPY-induced phosphorylation, but expression of GRK2(K220R) failed to attenuate the response (n = 2) (Fig. 5B). Furthermore, the protein kinase C inhibitor calphostin C (0.5 e) was unable to reduce agonist-stimulated Y1 receptor phosphorylation (n = 2) (Fig. 5B).
Constitutive Internalization of Truncated HA-Y1 Receptors. HAY1 receptor trafficking was investigated in the stable HEK293 clones, which provide a favorable cell type for the study of intracellular G protein-coupled receptor localization by immunofluorescence microscopy. In these experiments (performed at least three times independently), we identified only the receptor population that resided initially at the cell surface by labeling cells with anti-HA (8 e蘥/ml) before agonist treatment, fixation, and detection. Using this method, we observed no staining in nontransfected HEK293 cells. The addition of transferrin-Texas Red during agonist treatment identified the clathrin-coated internalization and subsequent endosomal pathways used by its constitutively recycling receptor.
Under basal conditions, HA-Y1 receptors were located predominantly at the plasma membrane in the HEK-HAY1 clone (Fig. 6), although we observed a relatively small degree of constitutive internalization of the receptor attached antibody in some cells. A rapid redistribution to punctate intracellular endosomes occurred on stimulation with NPY or PYY; significant HAY1 receptor internalization was observed at 10 nM agonist and for short incubation times of 5 min (1 e NPY; see Supplemental Fig. S1). Intracellular structures labeled with anti-HA antibody were distributed throughout the cytoplasm but became more concentrated close to the nucleus as agonist concentration or treatment time increased. Substantial, although not complete, colocalization with transferrin-Texas Red labeling was observed as illustrated in the example cell treated with 100 nM NPY in Fig. 6. BIBO3304 (1 e) did not alter HAY1 trafficking alone, but pretreatment with the antagonist blocked all internalization in response to NPY (Fig. 6). Preincubation with concanavalin A (0.3 mg/ml) prevented HAY1 receptor endocytosis and significantly restricted the cycling of the transferrin receptor, with punctate transferrin-Texas Red labeling predominantly at or close to the plasma membrane (data not shown).
In contrast to the cell-surface distribution of unstimulated HAY1 receptors, live labeling of either HEK-HAY1T or HEK-HAY1S cells with anti-HA resulted in substantial internalization of the antibody under basal conditions (Fig. 6); in each case, the pattern of intracellular localization and transferrin colocalization resembled that for HAY1 receptors stimulated by NPY but was unaffected by 1 e BIBO3304 (Fig. 6). In the continuous presence of concanavalin A, HAY1T and HAY1S receptors labeled with anti-HA were exclusively located at the plasma membrane (data not shown). We were surprised to find that the presence of agonist revealed differences in the behavior of HAY1T and HAY1S receptors. When stimulated with 100 nM NPY for the final 30 min of the HA antibody incubation, the predominantly intracellular location of HAY1T receptors did not change. However, significantly more HAY1S receptors were observed at the cell surface, and this redistribution was prevented by coincubation with 1 e BIBO3304 (Fig. 6).
Recruitment of GFP--Arrestin2 by HAY1 Receptors. We transiently expressed GFP-tagged -arrestin2 in the stable HEK-HAY1, HAY1T, and HAY1S cells to assess its distribution in unstimulated cells or those treated with NPY for different periods. Under control conditions, diffuse GFP fluorescence was observed throughout the cytoplasm in all three clones (Fig. 7). NPY stimulation (1 e) for 5 or 15 min resulted in the appearance of punctate endosomes intensely labeled with GFP in several HEK-HAY1 cells, but after 30-min agonist incubation, these structures were less prominent (Fig. 7A). A similar, although less pronounced, change in distribution was observed after NPY activation of the HAY1T receptor (Fig. 7B). However, no GFP-arrestin2 translocation could be observed in HAY1S cells under the same conditions (Fig. 7C).
Discussion
Here, we investigated the role of the Y1 receptor C terminus in regulating its G protein signaling, functional responses, and intracellular trafficking. We observed that truncation at Thr361 (HAY1T) or Ser352 (HAY1S) did not alter HA-tagged Y1 receptor G protein coupling, in contrast to a profound reduction in receptor efficacy after mutation of the more proximal palmitoylation site Cys337 (Holliday and Cox, 2003). However, desensitization of the HAY1S mutant was specifically inhibited, compared with either HAY1 or HAY1T receptors, using two complementary approaches. Only HAY1S responses (measured as NPY-stimulated [35S]GTPS binding in CHO clones) were substantially maintained after NPY pretreatment. In addition, stable expression of wild-type and truncated Y1 receptors in colony 1 epithelial layers enabled ISC measurement of their antisecretory responses in real time. Both full-length and truncated Y1 receptors exhibited an expected pharmacology, and loss of the distal C terminus did not cause apical misdirection of the Y1 receptor, in contrast to truncated luteinizing hormone and thyrotropin receptors (Beau et al., 2004). From our functional observations of sided responses, we cannot exclude an additional intracellular accumulation of poorly targeted S352 receptors, as observed for the truncated mGluR7a subtype (McCarthy et al., 2001). C1Y1S PYY responses, as an inhibition of VIP-stimulated ISC, remained strikingly sustained in the presence of maximal PYY concentrations in contrast to the transient nature of those from C1Y1 and C1Y1T clones. We therefore confirmed the effects of S352 truncation on desensitization in an assay in which the sequential processes of activation and inactivation can be followed after a single agonist addition (Holliday and Cox, 2003). Furthermore, these functional responses represent the culmination of a signaling cascade involving Gi-mediated decreases in cAMP production and a reduction in protein kinase A-sensitive basolateral K+ and apical CleC conductances (MacVinish et al., 1993), for which desensitizing mechanisms may exist at several stages [e.g., stimulation of Gi GTPase activity by regulators of G protein signaling proteins (Roy et al., 2003)]. Our data clearly demonstrate that, as the first step of this pathway, Y1 receptor inactivation is key in determining the pattern and duration of the emerging functional response.
We have demonstrated for the first time that Y1 receptors are phosphorylated in response to agonist and have shown that S352 but not T361 truncation inhibited this effect. Although Y1 receptor signaling can lead to protein kinase C activation, a specific inhibitor (calphostin C) did not inhibit its phosphorylation. Moreover, the protein kinase C recognition sites in the Y1 receptor C terminus (S362; Fig. 1) and third intracellular loop (T258) lie outside the critical phosphorylated sequence of four serine/threonine residues between Ser352 and Lys360 (Fig. 1), suggesting that the role of this kinase is limited in homologous Y1 receptor desensitization. On the other hand, our results clearly demonstrate that the 7TM receptor kinase GRK2 can phosphorylate the Y1 receptor. Given that the GRK2 K220R mutant did not prevent agonist-induced responses, an additional contribution of other GRK family members (particularly those that are constitutively membrane-associated) (Willets et al., 2003) is also possible.
Prior phosphorylation of agonist-occupied receptors by GRKs is a requirement for -arrestin recruitment, whose specificity is generated by dual domains that recognize the active conformation and phosphorylated sequences (Gurevich and Gurevich, 2004). Several investigations have implicated a short phosphorylated serine/threonine-rich stretch of the 7TM receptor C terminus in controlling -arrestin binding and desensitization, with recent examples including rhodopsin (Kisselev et al., 2004), EP4 prostaglandin (Neuschafer-Rube et al., 2004), and complement C5a receptors (Braun et al., 2003). Despite the relatively promiscuous nature of the -arrestin recognition domain (Gurevich and Gurevich, 2004), our results also suggest that phosphorylation of the short region between Ser352 and Lys360 is a minimum requirement for subsequent Y1 receptor desensitization. However, they do not exclude the possibility of sites in the more distal C terminus or those in the intracellular loops for which earlier phosphorylation of this region is required (Ohguro et al., 1993).
The involvement of -arrestin2 in Y1 receptor desensitization was originally suggested by Berglund et al. (2003), who measured the interaction between the two proteins on NPY stimulation in bioluminescence resonance energy transfer experiments (association t1/2 of 3eC5 min). We also observed that activated HAY1 and HAY1T but not HAY1S receptors caused the translocation of cytoplasmic GFP--arrestin2 to intracellular endosomes. This provides a consistent mechanism (inhibition of phosphorylation and -arrestin recruitment) for the specific effect of S352 truncation on desensitization in CHO and epithelial cells.
The internalization of NPY-stimulated HAY1 receptors in HEK293 cells is in accordance with previous studies using GFP-tagged Y1 receptors (Gicquiaux et al., 2002) or radiolabeled agonists (Parker et al., 2001; Pheng et al., 2003). As before, the speed of Y1 receptor endocytosis in response to NPY or PYY, colocalized with labeled intracellular transferrin receptors, indicated a mechanism dependent on clathrin-coated pits and the subsequent sorting of receptors to recycling endosomes. However, BIBO3304 did not alter Y1 receptor distribution (predominantly cell surface) in these experiments, beyond its effective blockade of NPY-induced endocytosis. The Y1 subtype is one of the few reported 7TM receptors to undergo antagonist-induced sequestration (Pheng et al., 2003), but it seems that this may either be specific for one compound (GR231118) or it reflects the very modest basal internalization that we observed.
It is surprising that unstimulated HAY1S and HAY1T receptors underwent substantially enhanced constitutive endocytosis, at least partly to transferrin-positive recycling compartments. Constitutive internalization of 7TM receptors can occur in the absence of agonist-independent activation (1a-adrenoceptor) (Morris et al., 2004) or as a consequence of it (CB1 and US28 receptors) (Fraile-Ramos et al., 2003; Leterrier et al., 2004). Although basal activation and endocytosis of a truncated sst2 somatostatin receptor has also been reported (Schwartkop et al., 1999), our observations indicate that Y1 receptor truncation mutants are not constitutively active. In contrast to the findings of Schwartkop et al. (1999), HAY1S and HAY1T receptors did not exhibit increased agonist affinities when expressed in any cell lines or a markedly enhanced sensitivity of agonist binding to GTPS. Neither truncation mutant elevated basal G protein activation in the [35S]GTPS assay, and the slight negative agonist activity of BIBO3304 was similar in both HAY1 and HAY1S membranes. In addition, basal internalization of the truncated HAY1 receptors was prevented by concanavalin A but not BIBO3304, and was -arrestineCindependent, suggesting a mechanism that does not require a spontaneously active phenotype.
Direct interaction between the HAY1T and HAY1S receptors and the AP-2 complex, which may underlie constitutive US28 receptor endocytosis in cells deficient in -arrestins1 and -2 (Fraile-Ramos et al., 2003), would provide a feasible alternative for their recruitment to clathrin-coated pits. YXX motifs recognized by the e?-adaptin subunit of AP-2 are present in both the third intracellular loop and the C terminus of the Y1 receptor (Fig. 1) and would be retained after S352 truncation. An equivalent C-terminal sequence has been implicated in the tonic internalization of the thomboxane A2 receptor (Parent et al., 2001) and in the thrombin-activated endocytosis of protease-activated receptor PAR1 (Paing et al., 2004), both of which are -arrestineCindependent. It is interesting that Paing et al. (2004) also observed that a PAR1 truncation just distal to the critical YXX motif (as for the HAY1S receptor) generated constitutive endocytosis. A similar consequence for HAY1S and HAY1T receptors may result from exposure of this motif and/or the loss of more distal inhibitory elements [e.g., those that may bind PDZ motif proteins (Fig. 1)] (Xiang et al., 2002). The contribution of this mechanism to tonic internalization of the full-length protein (which was low in HEK293 cells) could also be enhanced in cell types containing a high proportion of intracellular endogenous Y1 receptors (Zhang et al., 1999). This would generate a receptor pool protected from agonist exposure and would potentially allow more rapid resensitization (Parent et al., 2001).
We found that the absence of -arrestin2 binding to HAY1S receptors did alter its trafficking, because substantial redistribution to the plasma membrane in the presence of agonist distinguished the behavior of these receptors from the continued internalization of the HAY1T mutant. Constitutive endocytosis of HAY1S receptors may be retarded upon NPY stimulation by interaction of the active conformation with other plasma membrane proteins, perhaps those that organize G protein signaling complexes [e.g., a kinase-anchoring protein (Tao et al., 2003) or ezrin (Sitaraman et al., 2002)]. In this case, one role of -arrestin binding would be to dissociate such interactions (as required for desensitization), allowing endocytosis of agonist-occupied HAY1 and HAY1T receptors to proceed.
In summary, we have identified a C-terminal motif of the Y1 receptor that is the minimal requirement for its phosphorylation and is necessary for -arrestin2 binding and its rapid desensitization. Complexity in the trafficking of HAY1S and HAY1T receptors suggest the contribution of additional -arrestineCindependent mechanisms to the control of internalization, and future investigations to this end will shed new light on the additional protein interactions that regulate Y1 receptor signaling.
Acknowledgements
We thank Professor Roger Morris and Dr. Angela Jen for the use of the fluorescence microscope and excellent advice and Katie Gwilt for technical assistance. Dr. Jon Willets and Professors Steve Nahorski, Thue Schwartz, and Annette Beck-Sickinger kindly provided the cDNAs and antibodies used in this study.
doi:10.1124/mol.104.006114.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material..
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