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MSD Cardiovascular Research Center and Institute for Surgical Research, Rikshospitalet University Hospital, University of Oslo, Norway (J.D.P., T.A., H.K.J., H.A.)
Institute of Pathology, Rikshospitalet University Hospital, University of Oslo, Norway (M.P.O., H.S.H.)
Department of Pharmacology, Institute of Medical Biology, University of Troms, Norway (K.K., S.G.D.)
Abstract
We have previously reported that endocytic sorting of ETA endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxyl-terminal recycling motif of the ETA receptor. Assay of truncation mutants of the ETA receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 to 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel -strand structures (-finger). One of these segments was the -finger motif of neuronal nitric-oxide synthase reported to function as an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domain-binding ligand. Based on these findings, the three-dimensional structure of the recycling motif of ETA receptor was predicted to attain a -finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative -finger conformation or PDZ ligand function prevented recycling of the ETA receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxylterminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ETA receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.
The physiological effects of the vasoactive peptide endothelin-1 (ET-1) are mediated by the ETA and ETB receptors, which belong to class A G protein-coupled receptors (GPCRs) (Yanagisawa et al., 1988). In the vasculature, ETA receptors residing on smooth muscle cells mediate prolonged vasoconstriction, whereas ETB receptors, which are located on the plasma membrane of endothelial cells, are primarily considered to cause NO-mediated vasodilation (Yanagisawa and Masaki, 1989). In addition, considerable evidence now also supports a role for the ETB receptor in clearance of plasma ET-1 from the circulation (Berthiaume et al., 2000; Opgenorth et al., 2000). We have previously shown that agonist-induced internalization of the two receptor subtypes depends on a mechanism involving G protein-coupled receptor kinase, arrestin, clathrin, and dynamin (Bremnes et al., 2000). After internalization, however, the two receptor subtypes are targeted to different intracellular fates. The ETA receptor follows the recycling pathway through the pericentriolar recycling compartment and subsequently reappears on the plasma membrane, whereas the ETB receptor is directed to lysosomes for degradation (Bremnes et al., 2000). In terms of physiological effects, rapid recycling of the ETA receptor may provide basis for re-establishment of the signaling response and thus for the sustained vasoconstriction mediated by this receptor.
A key event of endocytic transport of signaling receptors is their sorting to either divergent recycling or degradative membrane pathways. The prevailing model proposes that internalized receptors are prevented from recycling by becoming sequestered and retained in multivesicular bodies (Sorkin and von Zastrow, 2002). According to this model, recycling is considered the default destiny of membrane receptors not being targeted to lysosomes. The model is based mainly on studies of endocytic trafficking of the transferrin receptor and the epidermal growth factor receptor (Mukherjee et al., 1997). Rapid recycling of these receptors has been reported even after all cytoplasmic residues have been removed (Mayor et al., 1993). This passive view of recycling has recently been challenged, at least as far as GPCRs are concerned. Emerging evidence, including a previous report from our laboratory (Paasche et al., 2001), indicates that recycling of GPCRs is dependent on signals residing in the cytoplasmic carboxyl-terminal region (Cao et al., 1999; Trejo and Coughlin, 1999; Kishi et al., 2001; Hirakawa et al., 2003; Tanowitz and von Zastrow, 2003). Analysis of carboxyl-terminally truncated ET receptors and ETA/ETB chimera revealed that lysosomal trafficking seems to be the "default" pathway without any requirement for cytoplasmic carboxyl-terminal sorting signals (Paasche et al., 2001). In contrast, recycling of the ETA receptor was dependent on a signal residing in the carboxyl-terminal tail of this receptor subtype. So far, however, the structural characteristics of a motif involved in sorting of a GPCR to the recycling pathway has been determined for the 2 adrenergic receptor (2-AR) only (Cao et al., 1999). The carboxyl-terminal end of the 2-AR contains a structure or sequence pattern (DSSL) typically recognized by class 1 PDZ (postsynaptic density 95/disc-large/zona occludens) domain proteins (Hall et al., 1998). The PDZ ligand of the 2-AR has been reported to bind EBP50/Na+/H+ exchange regulatory factor 1 [Ezrin-radixin-moesin (ERM)-binding phosphoprotein-50] and to promote recycling of the 2-AR by linking the receptor to the actin cytoskeleton through the ERM-binding domain of EBP50 (Hall et al., 1998; Cao et al., 1999). Furthermore, when grafted onto the carboxyl-terminal end of the -opioid receptor, this four-residue sequence (DSLL) was shown to re-route endocytosed -opioid receptor from the degradative to the recycling pathway (Gage et al., 2001). On the other hand, the carboxyl-terminal PDZ ligand of the 1-AR does not seem to be critical for recycling of this receptor subtype (Xiang and Kobilka, 2003). Furthermore, many receptors that are efficiently sorted to the recycling pathway, including the ETA receptor, lack classic carboxyl-terminal PDZ binding motifs (Trejo and Coughlin, 1999; Bremnes et al., 2000; Kishi et al., 2001; Hirakawa et al., 2003; Tanowitz and von Zastrow, 2003). Yet for several of these receptors, the cytoplasmic carboxyl-terminal region has been shown to provide signals critical for sorting of the receptors to the recycling pathway (Trejo and Coughlin, 1999; Bremnes et al., 2000; Kishi et al., 2001; Hirakawa et al., 2003; Tanowitz and von Zastrow, 2003). Thus, the aim of the present study was to identify and characterize the structural motif in the carboxyl-terminal tail of the ETA receptor that dictates recycling of the receptor. Bioinformatics and mutational analysis of ETA receptor sorting provided several lines of evidence that an internal PDZ ligand of the carboxyl-terminal region targets the receptor to the recycling pathway. Furthermore, additional evidence suggests that internal PDZ ligands may represent a more general principle involved in sorting of many G protein-coupled receptors.
Materials and Methods
Plasmid Constructs. Subcloning of human transferrin receptor cDNA into pcDNA1 and of the open reading frame of human ETA receptor into the mammalian expression vector pcDNA3 (pcDNA3-ETA) has been described previously (Bremnes et al., 2000). The hemagglutinin (HA) epitope-tagged, dominant-negative EBP50 truncation mutant EBP50ERM in pcDNA3 was generously provided by Dr. Mark von Zastrow (University of California, San Francisco) (Cao et al., 1999).
Construction of ETA Receptor Truncation Mutants. Truncation mutants of the ETA receptor with deletions of the cytoplasmic carboxyl-terminal tail were constructed in pcDNA3 by PCR-directed mutagenesis. The truncation mutants ETA417, ETA406, ETA401, ETA398, ETA394, and ETA390 were made by replacement of Asp418, Trp407, Gly402, Pro399, Met395, and Ser391, respectively, by stop codon and introduction of an XbaI restriction site downstream of the stop codon. The 3'-prime EcoRI-XbaI fragment of wild-type ETA (pcDNA3-ETA) was replaced by the corresponding EcoRI/XbaI-digested PCR product to introduce the deletions indicated above. The cDNA constructs were verified by DNA sequence analysis using the dideoxy chain termination method. Construction of ETA receptor with in-frame carboxyl-terminal fusion of green fluorescent protein (ETA-GFP) in the mammalian expression vector pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA) has been described previously (Bremnes et al., 2000). ETA406-GFP and ETA401-GFP (i.e., ETA receptor truncation mutants with in-frame fusion of GFP at the carboxyl-terminal truncation site) were made by introduction of a BamHI restriction site at the terminal amino acid in pcDNA3-ETA406 and pcDNA3-ETA401 using PCR-directed mutagenesis. The EcoRI/BamHI1 fragments of these modified vectors were subsequently transferred to EcoRI/BamHI-digested pEGFP-N1-ETA-GFP to generate pEGFP-N1-ETA406-GFP and pEGFP-N1-ETA401-GFP, respectively.
Construction of ETA Receptor Point Mutants. ETA receptors with point mutations in the cytoplasmic carboxyl-terminal tail were constructed by site-directed circular mutagenesis using pcDNA3-ETA as template and two complementary oligonucleotide primers harboring the desired mutation. The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) for circular mutagenesis was employed, and mutants were selected by DpnI and verified by DNA sequence analysis using the dideoxy chain termination method.
Cell Lines and Transfection. CHO-K1 cells (American Type Culture Collection, Manassas, VA) were maintained, propagated, and transfected as described previously (Bremnes et al., 2000; Paasche et al., 2001). For fluorescence microscopy experiments, cells were plated onto glass coverslips before transfection. For cytosolic Ca2+ determination, CHO cells were grown within a 10-mm diameter polyethylene cylinder attached to the central part of a glass coverslip.
Kinetics of 125I-ET-1 Internalization and Recycling. Assay of internalization and externalization of wild-type ETA receptor and the ETA receptor mutants were performed using receptor labeling with 125I-ET-1 (Amersham Biosciences, Piscataway, NJ) as described previously (Bremnes et al., 2000; Paasche et al., 2001). 125I-ET-1 binding to ETA receptor is essentially irreversible, and the sum of surface-bound and internalized 125I-ET-1 remained constant during the assay. Thus, analysis of the fraction of receptors remaining intracellularly or on the surface of the plasma membrane as function of time enabled assay of receptor recycling as previously validated (Paasche et al., 2001).
Radioligand Binding. Radioligand binding analysis of membrane preparations of transiently transfected CHO cells was performed according to Elshourbagy et al. (1993) with minor modifications (Bremnes et al., 2000).
Loading of CHO Cells with Fura-2 Acetoxymethyl Ester and Measurement of Cytosolic Ca2+ in Single Cells. Transiently transfected CHO cells on glass coverslips were washed once with Hanks' balanced salt solution (HBSS) and incubated in HBSS containing 5 e Fura-2 and 0.025% Pluronic F127 for 30 min at 37°C. After incubation, the cells were washed once and then incubated at 37°C for 30 min before assay of cytosolic Ca2+ concentrations in response to stimulation with ET-1. The methods and software for data acquisition and analysis of cytosolic Ca2+ concentrations in single cells have been described previously (Rttingen et al., 1995). In brief, the cells were illuminated at the excitation wavelengths of 345 nm and 385 nm 2.5 times per second. Fluorescent images were collected by a charge-coupled device video camera, stored on a videotape, and simultaneously digitized by a framegrabber controlled by a computer. Cytosolic Ca2+ concentrations were calculated from the ratio between fluorescence intensities at excitation wavelengths of 345 and 385 nm at user-selected squares covering single cells. After recording of baseline fluorescence, ET-1 was added (100 nM), and collection of fluorescent images was continued to measure agonist-stimulated Ca2+ levels.
Phosphoinositide Hydrolysis. ET-stimulated inositol phosphate generation was assayed in transfected CHO cells metabolically labeled with 3 e藽i [myo-3H]inositol as described previously (Paasche et al., 2001).
Western Blot Analysis. To verify expression of EBP50ERM, the HA epitope-tagged carboxyl-terminal truncation mutant of EBP50 (Cao et al., 1998), Western blot analysis of samples from untransfected and transiently transfected CHO cells was performed. The immunoblots were processed as described previously (Bremnes 2000), using anti-HA IgG2a and, subsequently, horseradish peroxidase-conjugated anti-mouse IgG and the enhanced chemiluminescence system (Amersham Biosciences) for detection of immunoreactivity.
Analysis of Intracellular Trafficking of Receptor-GFP Fusion Proteins by Confocal Laser Scan Microscopy. For analysis of ET receptor trafficking, transfected CHO cells attached to glass coverslips were placed on ice, washed, and preincubated with cycloheximide (10 mg/ml) in HBSS for 30 min to prevent accumulation of newly synthesized ETA-GFP in the Golgi apparatus. The cells were subsequently incubated with ET-1 (100 nM) for1 h at 4°C in binding buffer (HBSS containing 20 mM HEPES pH 7.4, 0.2% bovine serum albumin, 0.1% glucose, and 10 mg/ml cycloheximide). Endocytosis of agonist-stimulated ETA-GFP or GFP-tagged ETA receptor mutants was initiated by rapidly changing the cell medium to 37°C. The cells were incubated for various periods of time at 37°C to investigate the intracellular trafficking pathways of the different receptor mutants. The cells were subsequently fixed in 4% paraformaldehyde before mounting onto object slides using Mowiol (Hoechst). The cells were examined with a Leica TCS SP confocal microscope equipped with an Ar (488 nm) and two He/Ne (543 and 633 nm) lasers (Leica, Wetzlar, Germany). A plan-apochromat 100x/1.4 numerical aperture oil immersion objective was used. Multilabeled images were acquired sequentially with a charge-coupled device camera. Images were processed and overlaid using Photoshop 7.0 (Adobe Systems, Mountain View, CA).
Uptake of Red Fluorescent Transferrin and LDL. Loading of the pericentriolar recycling compartment with 25 e/ml tetramethylrhodamine-conjugated transferrin (Tf-Rhod; Molecular Probes, Eugene, OR) was performed in CHO cells cotransfected with ET receptor-GFP cDNA and cDNA for the human transferrin receptor as described previously (Bremnes et al., 2000). For labeling of lysosomes, DiI-LDL (Molecular Probes) was bound to the cells simultaneously with ET-1 at 4°C. Internalization of the agonist-bound receptor was subsequently performed at 37°C for 60 min, allowing receptor-bound LDL to enter lysosomal compartments. The cells were fixed in 4% paraformaldehyde before mounting of the samples and investigation by confocal laser scan microscopy.
Structure Predictions of the Cytoplasmic Carboxyl-Terminal Region of the ETA Receptor. The ICM Pro 3.0 program (Molsoft L.L.C., La Jolla, CA) was used for modeling and computer graphics visualizations (Abagyan and Totrov, 1994). Using a crystal structure of bovine rhodopsin as structural template (Palczewski et al., 2000), a three-dimensional model of the human ETA receptor (SwissProt no. P25101 ) was constructed. To obtain evidence of a structural motif within the cytoplasmic carboxyl-terminal region of the ETA receptor, the Protein Data Bank of The Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org/pdb/) was searched for proteins with sequence similarity to the Gln390 to Asn427 segment of the ETA receptor. The National Center for Biotechnology Information BLAST software (Altschul et al., 1997) maintained by the Swiss Institute of Bioinformatics (http://us.expasy.org/tools/blast/) was employed to identify proteins in the PDB database with amino acid sequence similarities to the Gln390 to Asn427 segment of the ETA receptor. The structures of any identified proteins were retrieved and visualized computer graphically using the ICM software.
Probing The Cytoplasmic Carboxyl-Terminal Region of Mammalian GPCRs for Internal PDZ Interaction Motifs. Amino acid sequences for 300 different mammalian family A GPCRs excluding the olfactory receptors, 15 different family B GPCRs and 22 different family C GPCRs were obtained from the SwissProt and TrEMBL protein sequence databases (Abagyan and Totrov, 1994). The ClustalX 1.82 protein analysis program (http://www-igbmc.u-strasbg.fr/BioInfo/) was first used to obtain crude alignments of these sequences. The resulting alignments were further edited and analyzed with the BioEdit computer program. Thereafter, the carboxyl-terminal regions were retrieved from these alignments, starting from the positions corresponding to Val372 of the human ETA receptor (family A), Glu398 of the human secretin receptor (family B), and Glu865 of the human GABAB1 subunit (family C). The ICM 3.0 program was employed to search for putative internal PDZ ligand motifs in the carboxyl-terminal tail sequences using the following amino acid sequence pattern {[]\5}[ST][]{[]\3eC6}[ST]{[]\4}{[]\3}. {[]\n} represents any n consecutive amino acids. [ST] represents either a serine or a threonine residue. The aminoterminal [ST] of the sequence pattern corresponds to the PeC2 position of the internal PDZ pseudopeptide. represents a hydrophobic residue (P0 position) (i.e., isoleucine, valine, leucine, methionine, or phenylalanine). represents any positively charged residue (i.e., arginine, lysine, or histidine).
Results
Functional Characterization of Wild-Type ETA Receptor, ETA Receptor Mutants, and ETA-GFP. Stimulation of CHO cells transfected with either wild-type ETA receptor or the truncation mutants ETA406 or ETA401 with ET-1 (100 nM) generated rapid elevations in intracellular calcium concentrations (Fig. 1A). Untransfected cells, however, did not respond to stimulation with ET-1. Peak calcium concentrations seemed to be slightly higher in cells transfected with the ETA406 or ETA401 truncation mutants than in ETA wild-type transfected cells, but these differences were not statistically significant. Thus, the ETA receptor mutants were found to have similar capacities for generation of second messenger responses as their wild-type counterpart. In-frame carboxyl-terminal fusion of GFP was employed to investigate the intracellular trafficking pathways of ETA receptor. As demonstrated in Fig. 1B, internalization of 125I-ET-1 in CHO cells transfected with ETA-GFP showed that internalization of agonist-bound ETA-GFP proceeded with similar kinetics as that of wild-type ETA receptor. Furthermore, the capacity of ETA-GFP to mediate ET-1-stimulated inositol phosphate generation, as shown in Fig. 1C, was nearly identical to that of wild-type ETA receptor. Agonist-induced endocytosis of ETA-GFP was also examined with confocal laser scan microscopy. CHO cells transfected with ETA-GFP were incubated with ET-1 (100 nM) for 1 h at 4°C. Unbound ligand was subsequently removed and endocytosis of agonist-bound receptor was initiated by changing of the medium to 37°C. As seen in Fig. 1D, ETA-GFP is initially localized predominantly at the plasma membrane. However, within minutes after agonist-induced endocytosis of ETA-GFP, the receptor was detected in a spot-like structure near the nucleus demonstrating agonist-dependent intracellular transport. Figure 1E demonstrates endocytosis and intracellular trafficking of Tf-Rhod in CHO cells transfected with human transferrin receptor. As shown, Tf-Rhod seems to be rapidly internalized and transported through endosomal vesicles to a perinuclear endosomal compartment. After 15 min, Tf-Rhod is almost entirely located in the spot-like perinuclear structure previously demonstrated to represent the perinuclear recycling compartment (Presley et al., 1993). Therefore, for optimal labeling of the recycling compartment in subsequent experiments, agonist-dependent endocytosis was allowed to proceed for 15 min.
Intracellular Trafficking of ETA Receptor Mutants with Deletions of the Cytoplasmic Carboxyl-Terminal Tail. To identify the domain in the carboxyl-terminal tail of the ETA receptor that mediates recycling, a series of deletion mutants were constructed by introducing stop codons at positions Asp418 (ETA417), Trp407 (ETA406), Gly402 (ETA401), Pro399 (ETA398), Met395 (ETA394), or Ser391 (ETA390). The ligand binding characteristics of these truncated receptors did not differ significantly from that of wild-type ETA receptor (Table 1). As shown in Fig. 2, the ETA406 truncation mutant showed a biphasic internalization curve similar to that of wild-type ETA receptor. The declining part of this curve pattern (representing increasing fractions of receptor reappearing at the surface of the plasma membrane) has previously been shown to reflect recycling of the ETA receptor (Paasche et al., 2001). The ETA417 mutant (data not shown) also retained a biphasic internalization curve and hence the capability to undergo recycling. For the ETA receptor mutants with truncations amino-terminal to Gln406 (ETA401, ETA398, ETA394, ETA390), however, the time course of internalization shifted from a biphasic to a monophasic event, as shown in Fig. 2. We have previously shown that the signal for recycling resides distal to amino acid Gln390 of ETA (Paasche et al., 2001). Thus, the current observations provide evidence that the amino acid sequence 390 to 406 contains the critical information conferring the ability of the receptor to undergo recycling. To further investigate the intracellular trafficking pathways of the truncation mutants ETA406 or ETA401, GFP was fused to the carboxyl-terminal end of these receptor mutants for analysis of subcellular localization using fluorescence microscopy. ETA406-GFP and ETA401-GFP were transiently transfected into CHO cells and subjected to analysis of agonist-stimulated internalization. The GFP-tagged wild-type ETA receptor (ETA-GFP) was analyzed in parallel for comparison. As shown in Fig. 3, after 15 min of ET-1 induced internalization, both ETA-GFP and ETA406-GFP accumulated in a perinuclear spot-like structure that was colabeled with red fluorescent transferrin (i.e., a prototypical marker of the perinuclear recycling compartment) (Presley et al., 1993). ET-induced internalization of ETA401-GFP, however, was associated with spread endosome-like vesicular structures throughout the cytoplasm with only minor colocalization in the recycling compartment. This finding indicates that the transport route of ETA401 differs from that of ETA-GFP and ETA406-GFP. To investigate whether the spread vesicular distribution of internalized ETA401 was consistent with transport to lysosomes, the lysosomal compartments of ETA406-GFP- or ETA401-GFP-transfected cells were labeled with red fluorescent LDL (DiI-LDL) (Handley et al., 1981). As shown in Fig. 4, neither ETA-GFP nor ETA406-GFP colocalized with DiI-LDL after 60 min of internalization. However, the spread endosome-like structures of internalized ETA401-GFP demonstrated extensive colocalization with DiI-LDL (Fig. 4).
The properties of ET-1 binding, including the number of binding sites, were determined in CHO cells transiently transfected with ETA wild-type, ETA-406, ETA-401, ETA-390, ETA-T396A S397A, ETA-T403A S404A, ETA-406-GFP, and ETA-401-GFP. Binding of 125I-ET-1 was performed on membranes as described under Materials and Methods to determine the equilibrium dissociation constants (Kd) and the maximal binding (Bmax) for the different receptors. The data are mean ± S.D. of three parallels and representative of at least three independent experiments.
Identification and Characterization of a Motif in the Carboxyl-Terminal Tail of the ETA Receptor That Dictates Receptor Recycling. Searching of the PDB database revealed that specific domains of rat CD2 (PDB code 1hng ), human interferon- receptor chain (IFN- R) (PDB code 1fyh ), and human neuronal nitric-oxide synthase (nNOS) (PDB code 1qau ) displayed significant similarities to the Gln390 to Asp411 peptide segment of ETA (Fig. 5, A and B). Residues Phe103 to Gln124 of human nNOS, Ile131 to Thr151 of human IFN- R, and Tyr129 to Asn149 of rat CD2 displayed 36, 40, and 40% amino acid similarity, respectively, to the Gln390 to Asp411 peptide segment of the human ETA receptor. Furthermore, the identified segments of CD2, IFN- R, and nNOS all contained antiparallel -strands adopting -finger structures. Indeed, the -finger of human nNOS has been reported to function as an internal PDZ-ligand (Christopherson et al., 1999). In this motif, the proximal -strand (also referred to as the pseudopeptide motif) structurally mimics distal carboxyl-terminal PDZ-ligands in its sequence-specific interaction with one of the PDZ domains of syntrophin. Analysis of the crystal structure of the PDZ-ligand of human nNOS in complex with the PDZ domain of syntrophin, revealed that the hydrophobic phenylalanine at the end of the proximal -strand (G-strand) is exposed by a sharp -turn and mimics the free carboxylate group normally required for the hydrophobic P0 position of a carboxyl-terminal ligand (Harris et al., 2001). Based on sequence similarities between the ETA receptor and the proteins shown in Fig. 5, A and B, and using the structure of nNOS as a template, a three-dimensional model of the Lys392 to His410 peptide sequence of the ETA receptor was built. In the predicted structure, as shown in Fig. 5C, the proximal -strand was found to contain a sequence conforming to Class 1 PDZ ligands with Val398 and Thr396 of ETA representing the P0 and the PeC2 positions, respectively, of the putative PDZ ligand.
Intracellular Trafficking of ETA Receptors with Mutations that Perturb the PDZ Recognition Sequence or Structural Integrity of the -Finger. To provide further evidence that the 390 to 406 peptide sequence of ETA possesses PDZ ligand properties, we used site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative ligand. Figure 6, A and B, show the kinetics of agonist-induced internalization of ETA wild-type and ETA receptor mutants with substitutions perturbing the proximal strand of the -finger (i.e., the strand containing the ligand directly interacting with a PDZ domain-containing protein). Agonist-stimulated internalization of the ETA T396A/S397A mutant was substantially impaired compared with its wild-type counterpart (Fig. 6A). Furthermore, the internalization curve was shifted from a biphasic to a monophasic event, which indicates that mutations of the PeC1 and PeC2 positions of the putative PDZ ligand prevent recycling. Mutations of V398, which represents the hydrophobic P0 position into alanine or glycine (Fig. 6B) did not affect the initial rate of internalization but abolished the biphasic curve pattern, indicating severely perturbed recycling. To test our structural model further, mutations at residues that would perturb the distal strand of the -finger were also made. As shown in Fig. 6, C and D, agonist-induced internalization of the ETAT403A/S404A mutant exhibited a monophasic internalization curve similar to that of the ETAT396A/S397A mutant, where both the initial rate of internalization and recycling capabilities were affected. The I405A and I405G mutations had less dramatic albeit similar effects as V398A and V398G mutations. Inhibition of recycling was more pronounced upon mutation to alanine but was observed for both the ETAI405A and ETAI405G mutants (Fig. 6D). To study the intracellular trafficking pathways of these point-mutated receptors, GFP fusion proteins of ETAT396A/S397A and ETAT403A/S404A mutants were made. Agonist-stimulated internalization of receptor was investigated together with simultaneous uptake of red fluorescent Tf-Rhod or DiI-LDL as described under Materials and Methods. As shown in Fig. 3, neither the ETAT396A/S397A-GFP nor ETAT403A/S404A-GFP mutants colocalized with transferrin receptor in the pericentriolar recycling compartment as opposed to ETA-GFP. However, in cells transfected with ETAT396A/S397A-GFP or ETAT403A/S404A-GFP, labeled with DiI-LDL, and stimulated with ET-1 for 60 min, extensive colocalization of receptor and DiI-LDL was detected in lysosomes (Fig. 4). Taken together, these results support our hypothesis that recycling of ETA receptor is mediated via PDZ domain interactions.
Analysis of Thr396, Ser397, Thr403, and Ser404 of ETA as Potential Sites of Phosphorylation. As described above, substitutions of serine and threonine in the ETAT396A/S397S or ETAT403A/S404A double mutants affected not only the recycling capabilities but also the rate of internalization. Based on these observations, we examined whether the indicated threonine and serine residues were sites of phosphorylation by introducing negative charge in the form of an aspartate residue that could mimic a phosphorylated state in these positions. We constructed mutants of ETA406 and "full-length" ETA receptors in which Thr396, Ser397, Thr403, and Ser404 were substituted with aspartate. Mutations into alanine were also made for parallel comparison. As shown in Fig. 7, A and B, the aspartate and alanine substitutions of "full-length" ETA and ETA406 abolished the biphasic internalization kinetics and significantly reduced the rate of internalization in a near identical manner. Based on these observations, we conclude that receptor internalization and recycling are similarly affected upon mutation to alanine or aspartate.
Effect of a Dominant-Negative EBP50 Mutant on Agonist-Stimulated Internalization and Recycling of the ETA Receptor. To investigate whether or not recycling of the ETA receptor is dependent on EBP50, we used a dominant-negative truncation mutant of EBP50 (EBP50ERM) that lacks the ERM-binding domain but retains the PDZ domain reported to be required for interaction with the 2-AR (Hall et al., 1998). CHO cells were transfected with the ETA receptor alone or together with EBP50ERM. Western blot analysis, as shown in Fig. 8, confirmed the expression of EBP50ERM in the transiently transfected cells. The biphasic internalization kinetics of the ETA receptor, which indicates recycling, was not altered upon expression of EBP50ERM (Fig. 8).
Probing for Internal PDZ Ligand Motifs in GPCRs. A specific sequence pattern required to fulfill the structural criteria of an internal PDZ ligand (described under Materials and Methods) were defined and used to construct an algorithm that could be employed to screen GPCRs. Analysis of the carboxyl-terminal regions of more than 300 mammalian family A, B, and C GPCRs, starting after predicted endpoints of transmembrane helix 7, revealed 35 different human GPCRs that fulfilled the sequence criteria of an internal PDZ ligand motif. Figure 9 shows alignment of the identified carboxyl-terminal sequences of 27 human GPCRs identified in the above analysis for which the cognate ligand is known and eight orphan GPCRs.
Discussion
In the present study, the carboxyl-terminal motif of ETA receptor that dictates receptor recycling has been identified and characterized. Structural modeling and mutagenesis of the cytoplasmic carboxyl-terminal region of ETA receptor revealed that receptor recycling is mediated by a motif with the structural characteristics of an internal PDZ ligand.
Composite data from studies of ETA and ETB receptor chimera in which the carboxyl-terminal tail of the receptors were interchanged (Paasche et al., 2001), and from studies of increasing truncations of the carboxyl-terminal region of ETA, revealed that the 17 amino acid polypeptide segment Gln390 to Gln406 of ETA receptor constitutes the minimal peptide sequence required to provide a functional signal for recycling. It is surprising that the identified polypeptide segment was part of a slightly larger segment (Gln390eCAsp411) of the carboxyl-terminal region of ETA that displayed striking similarities to peptide sequences of nNOS, IFN- R, and CD2 for which a common secondary and tertiary structure had been determined. The shared structure of the latter proteins consisted of antiparallel -strands forming -fingers that in nNOS have been reported to function as a PDZ ligand (Hillier et al., 1999). Although the crystal structure of the ETA receptor has not yet been determined, prediction of the secondary and tertiary structures of the Gln390 to Asp411 polypeptide segment of ETA receptor revealed that this segment might also consist of antiparallel -strands attaining -finger conformation. The proximal and distal -strands of the putative -finger could be supported by several hydrogen bonds between residues within the Gln390 to Asp411 polypeptide segment, one of which was a hydrogen bond typically found between the first and the fourth residues of the loop between the -strands. The latter is considered crucial in stabilizing the -turn (Coligan et al., 2004). Additional lines of evidence also reported in this study consistently supported involvement of the putative -finger of ETA in receptor recycling. Targeted mutations of residues within the proximal -strand (ETAT396A/S397A, ETAV398A, ETAV398G, ETAT403A/S404A, ETAI405A, ETAI405G) or distal -strand (ETAT403A/S404A, ETAI405A, ETAI405G) all disrupted recycling of ETA receptor. These observations indicate that mutations that affect the structural integrity of the -finger abolish receptor recycling. However, to provide unequivocal evidence of a PDZ-mediated mechanism of ETA receptor recycling, binding of the putative -finger and a PDZ-containing protein would have to be demonstrated and implicated a role in receptor recycling. It is striking that the proximal -strand of the putative -finger of ETA also contained a pseudopeptide sequence conforming to the canonical class I PDZ ligand similar to that of the proximal -strand of the nNOS -finger. According to the predicted -finger of ETA, Val398 represents the indispensable hydrophobic residue at the P0 position of the putative PDZ ligand. As demonstrated in the present study, alanine substitution of the PeC2, PeC1, or P0 residues of the PDZ pseudopeptide ligand in the proximal -strand essentially eliminated recycling of ETA receptor. These results correlate with the loss of function caused by alanine substitution of the corresponding PDZ pseudopeptide residues (T109A or F111A at the PeC2 and P0 positions, respectively) of nNOS (Harris et al., 2001).
Comparing the putative -finger of ETA receptor with that of nNOS also revealed conservation of a positive residue in the distal -strand. Arg121 in the distal -strand of nNOS forms a salt bridge with Asp62 and plays important role in binding of nNOS to the PDZ domain of syntrophin, presumably by stabilizing the -finger formation (Harris et al., 2001). The specific function of Lys408 in the putative -finger motif of ETA receptor (i.e., the positive residue aligning with Arg121 of the distal -strand nNOS) is unclear because this residue is removed in the ETA-406 truncation mutant, which is still capable of recycling. However, it has been reported that the -finger of nNOS is a fairly rigid structure that, even by itself, separate from nNOS, may retain some residual hairpin structure (Wang et al., 2000).
Previous reports have shown that agonist-stimulated desensitization and internalization of ETA and ETB receptors depend on GRK-catalyzed phosphorylation of serine and threonine residues in the cytoplasmic carboxyl-terminal region (Freedman et al., 1997; Bremnes et al., 2000). However, the specific residues of agonist-occupied ETA receptors phosphorylated by GRK have yet to be determined. It is conceivable that introduction of negative charge by phosphorylation of any serine or threonine residue of the carboxyl-terminal region may to some extent relieve intramolecular constraints sufficient to promote binding of -arrestin with subsequent desensitization and internalization of the receptor. However, mimicking of ETA receptor phosphorylation by substituting the indicated threonine and serine residues with aspartic acid (ETAT396D/S397D; ETAT403D/S404D) did not lead to increased rates of agonist-induced internalization. Rather, the aspartic acid substitutions impaired agonist-induced internalization to an extent similar to that of alanine substitution of the same residues. (ETAT396A/S397A; ETAT403A/S404A). Thus, the reduced initial rates of agonist-induced internalization of the ETA receptor mutants do not seem to be caused by loss of GRK-catalyzed phosphorylation. A more plausible interpretation of the observed findings is that substitution of the serine and threonine residues of the proximal and distal -strands disrupt the -finger and thereby abolish ETA receptor recycling. As serially connected mechanisms, impaired endosomal trafficking may also affect the initial step of endocytosis (i.e., agonist-induced internalization of the ETA receptor).
To our knowledge, this is the first demonstration implicating a putative internal PDZ ligand structure in endocytic sorting of a GPCR. An imminent issue then, was to what extent the putative internal PDZ ligand of ETA receptor might represent a more widely employed principal in endocytic sorting of GPCRs. It is interesting that as many as 35 different GPCRs fulfilled the principal criteria of an internal PDZ ligand motif. However, our search parameters only entailed class I PDZ domains. In this respect, inclusion of parameters covering class II and III PDZ domain proteins could potentially identify even more GPCR with internal PDZ ligand motifs. As shown in Fig. 9, the loop connecting the antiparallel -strands of the putative -finger structures of the aligned GPCRs consists of varying number of residues. Indeed, the variation in loop length allowed in the search parameters for -finger-like PDZ ligand motifs in GPCRs was supported by evidence of maintained PDZ binding affinity upon insertion of additional residues in the -turn region of the PDZ ligand of nNOS (Harris et al., 2001).
Among the human GPCRs shown in Fig. 9 are several receptors that have been reported to follow intracellular recycling pathways: the 1D-adrenergic, AT1 angiotensin, NK1 neurokinin, Y1 neuropeptide, luteinizing hormone (LH), 5-HT2A serotonin, SST3 somatostatin, and V1A vasopressin receptors (Benya et al., 1994; Tseng et al., 1995; Garland et al., 1996; Hein et al., 1997; Innamorati et al., 1999; McCune et al., 2000; Kishi et al., 2001; Kreuzer et al., 2001; Marchese and Benovic, 2001; Bhattacharyya et al., 2002; Chauvin et al., 2002; Gicquiaux et al., 2002). On the other hand, peptide sequences with similarity to the search parameters could not be identified in GPCRs sorting to lysosomes: the ETB receptor, PAR-1, CXCR4 chemokine receptor, -opioid receptor, and rat LH receptor (Trejo and Coughlin, 1999; Bremnes et al., 2000; Gage et al., 2001; Kishi et al., 2001; Marchese and Benovic, 2001). Thus, the proposed model of internal PDZ ligand recognition seems to be a consistent mechanism for sorting GPCRs to the recycling pathway. The rat and human LH receptor homologs deserve special attention because of divergent sorting to lysosomes and endocytic recycling, respectively (Kishi et al., 2001). It is interesting that the primary structure of rat LH receptor differs from its human receptor homolog at residues critical to maintain the putative PDZ ligand-containing -finger. Thus, rat LH receptor may be regarded as a naturally occurring variant of human LH receptor that does not contain an operative PDZ ligand. This contention is supported by several reports of point mutations or deletion of amino acids in the carboxyl-terminal region of human LH receptor that divert the receptor from the recycling pathway to the lysosomal trafficking pathway (Kishi et al., 2001; Hirakawa et al., 2003; Galet et al., 2004). Indeed, these targeted amino acids are apparently all critical to maintain the PDZ pseudopeptide ligand or -finger conformation according to our proposed model. A recent report by Ascoli and colleagues demonstrated that efficient recycling was also shown to be dependent on the upstream hydrophobic amino acid Leu683 (Galet et al., 2004). According to the alignment in Fig. 9, Leu683 may represent the "0-position" of a putative PDZ ligand-containing -finger formation.
Although sorting of GPCRs to the recycling pathway may depend on uniform mechanisms, different PDZ domain proteins may be involved. For example, involvement of EBP50 does not seem to be a consistent finding (Cao et al., 1999; Kishi et al., 2001). In the present study, a dominant-negative deletion mutant of EBP50 (EBP50), lacking the ERM binding domain, did not affect recycling of the ETA receptor despite massive expression of EBP50.
In the present study, bioinformatic and mutational analyses provide strong evidence that recycling of the ETA receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This motif, which is lacking in the carboxyl-terminal region of ETB endothelin receptors, provides a mechanism of the divergent sorting of the ETA and ETB receptor subtypes. Furthermore, we provide evidence that internal PDZ ligand motifs may represent a more general principle of endocytic sorting of GPCRs. The important challenge of the future will be to identify the protein that recognizes and binds the putative PDZ ligand motif of ETA receptor and directs the receptor to the recycling pathway.
doi:10.1124/mol.104.007013.
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