Role of the Differentially Spliced Carboxyl Terminus in Thromboxane A2 Receptor Trafficking
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m009375200
ISSN1083-351X
AutoresJean‐Luc Parent, Pascale Labrecque, Moulay Driss Rochdi, Jeffrey Benovic,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoThe thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not TPα, also undergoes tonic internalization. Tonic internalization of TPβ was temperature- and dynamin-dependent and was inhibited by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2φ or YX 3φ motif in the carboxyl-terminal tail of TPα induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPβ and represent the first report of a YX 3φ motif involved in tonic internalization of a cell surface receptor. The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not TPα, also undergoes tonic internalization. Tonic internalization of TPβ was temperature- and dynamin-dependent and was inhibited by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2φ or YX 3φ motif in the carboxyl-terminal tail of TPα induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPβ and represent the first report of a YX 3φ motif involved in tonic internalization of a cell surface receptor. G protein-coupled receptor thromboxane A2 receptor G protein-coupled receptor kinase phosphate-buffered saline enzyme-linked immunosorbent assay human embryonic kidney Dulbecco's modified Eagle's medium bovine serum albumin Tris-buffered saline hemagglutinin polymerase chain reaction Cell surface receptors provide a primary mechanism by which cells perceive their environment. Many cell surface receptors are dynamically regulated and often undergo a process of endocytic sorting (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar). For some receptors (e.g. G protein-coupled and growth factor), sorting is often initiated by hormone binding, whereas for others (e.g. low density lipoprotein and transferrin), the receptors undergo continuous or tonic internalization and recycling. Recent studies have demonstrated that several GPCRs1 including the CXCR4, thyrotropin, M2 muscarinic, and thrombin receptors also undergo tonic internalization (2Signoret N. Oldridge J. Pelchen-Matthews A. Klasse P.J. Tran T. Brass L.F. Rosenkilde M.M. Schwartz T.W. Holmes W. Dallas W. Luther M.A. Wells T.N.C. Hoxie J.A. Marsh M. J. Cell Biol. 1997; 139: 651-664Crossref PubMed Scopus (335) Google Scholar, 3Baratti-Elbaz C. Ghinea N. Lahuma O. Loosfelt H. Pichon Milgrom E. Mol. Endocrinol. 1999; 13: 1751-1765Crossref PubMed Scopus (70) Google Scholar, 4Roseberry A.G. Hosey M.M. J. Biol. Chem. 1999; 274: 33671-33676Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Although no particular motif responsible for tonic internalization of GPCRs has been identified, tyrosine-containing (YXXφ and NPXY) and dileucine motifs have been shown to be determinants for a number of other receptor types (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar). Various studies have demonstrated direct interaction between YXXφ motifs and the μ chain of the clathrin-associated proteins AP-1, AP-2 (Ref. 6Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar and references therein), and AP-3 (7Ohno H. Aguilar R.C. Yeh D. Taura T. Saito T. Bonifacino J.S. J. Biol. Chem. 1998; 273: 25915-25921Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 8Le Borgne R. Alconada A. Bauer U. Hoflack B. J. Biol. Chem. 1998; 273: 29451-29461Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), allowing the efficient targeting of transmembrane proteins containing these motifs to clathrin-coated vesicles.Thromboxane has been implicated in a number of cardiovascular, bronchial, and kidney diseases (9Halushka P.V. Allan C.J. Davis-Bruno K.L. J. Lipid Med. Cell Signal. 1995; 12: 361-378Crossref PubMed Scopus (50) Google Scholar, 10Spurney R.F. Middleton J.P. Raymond J.R. Coffman T.M. Am. J. Physiol. 1994; 267: F467-F478PubMed Google Scholar). It is produced by the sequential metabolism of arachidonic acid by cyclooxygenase and thromboxane synthase following activation of a variety of cell types including platelets, macrophages, and vascular smooth muscle cells (11Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). Thromboxane is a strong activator of platelet aggregation and smooth muscle cell proliferation and mediates its effects via interaction with a specific GPCR. The thromboxane A2receptor (TP) is encoded by a single gene that is alternatively spliced in the carboxyl terminus resulting in two variants, TPα (343 residues) and TPβ (407 residues) that share the first 328 amino acids (12Morinelli T.A. Halushka P.V. Trends Cardiovasc. Med. 1991; 4: 157-161Crossref Scopus (17) Google Scholar, 13Hirata M. Hayashi Y. Ushikubi F. Yokota Y. Kageyama R. Nakanishi S. Narumiya S. Nature. 1991; 349: 617-620Crossref PubMed Scopus (615) Google Scholar, 14Raychowdhury M.K. Yukawa M. Collins L.J. McGrail S.H. Kent K.C. Ware J.A. J. Biol. Chem. 1994; 269 (; Correction (1995) J. Biol. Chem.270, 7011): 19256-19261Abstract Full Text PDF PubMed Google Scholar).In a previous study, we demonstrated that TPβ, but not TPα, undergoes agonist-induced internalization in a variety of cell types (15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Internalization of TPβ was dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Additional characterization of the role of arrestins in this process revealed that arrestin-3 coexpression promoted agonist-induced internalization of both TPα and TPβ but not of a mutant truncated after residue 328. Analysis of various carboxyl-terminal deletion mutants revealed that a region between residues 355 and 366 in TPβ was essential for agonist-promoted internalization. During the course of these studies, we observed that TPβ, but not TPα, also undergoes tonic internalization. In the present study, we characterize the mechanisms involved in tonic internalization of TPβ. These studies reveal that a YX 3φ motif found in the proximal portion of the carboxyl-terminal tail of TPβ is responsible for tonic internalization.RESULTS AND DISCUSSIONA schematic representation of the carboxyl terminus of the two isoforms of the human thromboxane A2 receptor is shown in Fig. 1. To investigate tonic internalization of TPα and TPβ, epitope-tagged receptors were transiently expressed in HEK293 cells. Previous studies have shown that the agonist binding affinities of TPα and TPβ are similar (14Raychowdhury M.K. Yukawa M. Collins L.J. McGrail S.H. Kent K.C. Ware J.A. J. Biol. Chem. 1994; 269 (; Correction (1995) J. Biol. Chem.270, 7011): 19256-19261Abstract Full Text PDF PubMed Google Scholar, 15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 16Hirata T. Ushikubi F. Kakizuka A. Okuma M. Narumiya S. J. Clin. Invest. 1996; 97: 949-956Crossref PubMed Scopus (252) Google Scholar, 17Habib A. Vezza R. Créminon C. Maclouf J. FitzGerald G.A. J. Biol. Chem. 1997; 272: 7191-7200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Moreover, addition of a FLAG epitope at the amino terminus of the receptors did not alter ligand affinities, nor did it affect the activation characteristics of the receptors as determined by their respective EC50 values for agonist-promoted generation of inositol phosphate (data not shown).During our immunofluorescence analysis of agonist-induced internalization of thromboxane A2 receptors, we noted that TPβ could undergo tonic internalization (15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). To further characterize this phenomenon, we performed a series of immunofluorescence studies on cells transiently expressing FLAG-tagged TP receptors. Cells were initially incubated with the FLAG antibody at 4 °C for 1 h, washed, and then incubated at different temperatures so that tonic receptor internalization could be followed. As shown in Fig.2 A, there is significant redistribution of TPβ to intracellular compartments following incubation at 37 °C whereas minimal internalization of TPα is observed. Quantitation of intracellular and total cell fluorescence revealed that ∼60% of TPβ was tonically redistributed to a subcellular compartment following a 1-h incubation at 37 °C. These observations confirm that TPβ, but not TPα, undergoes significant tonic internalization. In contrast, when the cells are incubated for 1 h at 4 °C or 16 °C, TPα and TPβ remain entirely at the cell surface. Interestingly, the inability of TPβ to undergo tonic internalization at 16 °C is a property shared with agonist-induced internalization of TPβ (data not shown) and the β2AR (18Cao T.T. Mays R.W. von Zastrow M. J. Biol. Chem. 1998; 273: 24592-24602Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), but quite distinct from tonic internalization of the transferrin receptor which still occurs at 16 °C (data not shown) (18Cao T.T. Mays R.W. von Zastrow M. J. Biol. Chem. 1998; 273: 24592-24602Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar).Figure 2Immunofluorescence analysis of TP α and TP β distribution in HEK293 cells. A, FLAG-tagged receptors were transiently transfected in HEK293 cells. Cells were incubated with the FLAG antibody at 4 °C prior to any other treatment to detect receptors that were present initially at the cell surface. Immunofluorescence detection was performed as described under "Experimental Procedures." Top panel, receptor distribution in cells expressing TPα (left) and TPβ (right) when incubated at 4 °C. Middle panel, after a 1 h incubation at 16 °C. Bottom panel, after a 1 h incubation at 37 °C. B, HEK293 cells, transiently transfected with TPβ, were labeled with FLAG antibody at 4 °C prior to incubation at 37 °C for 1 h. Tonic internalization of TPβ in the presence or absence of the TP receptor antagonist SQ29548, the inhibitors of clathrin-coated pit formation NH4Cl and sucrose, or dynamin-K44A was analyzed by immunofluorescence and quantitated. Data represent the percentage of intracellular immunofluorescence relative to total immunofluorescence of individual cells. Immunofluorescence was measured using the NIH Image 1.62b7f software. Results shown represent the mean ± S.E. of three independent experiments, where immunofluorescence of at least ten cells was evaluated for each experiment. Refer to "Experimental Procedures" for details.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To verify that the internalization of TPβ was not triggered by endogenous production of thromboxane or by trace amounts of agonist in the media, cells were pretreated with the TP antagonist SQ29548 (10 μm) or the cyclooxygenase inhibitor indomethacin (10 μm) for 15 min at 4 °C prior to incubation at 37 °C. These treatments had no effect on internalization of TPβ confirming that the receptor is undergoing tonic internalization (Fig.2 B). The same trafficking properties for TPα and TPβ were observed in P388D1 and A431 cells, which express endogenous TP receptors, and Mia Paca, COS-1 and CHO cells, which lack endogenous TP receptors, as well as in HEK293 cells stably expressing low levels of thromboxane receptors (15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) (data not shown). These results demonstrate that the differential tonic internalization of TPα and TPβ is a property of the receptor.We previously demonstrated that agonist-induced internalization of TPβ is dynamin-, GRK2-, and arrestin-dependent (15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Thus, we next determined the role of these proteins in tonic internalization of TPβ. Immunofluorescence analysis of TPβ redistribution in the presence of dominant-negative mutants of dynamin (19Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar), GRK2 (20Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar), and arrestin-3 (21Orsini M.J. Benovic J.L. J. Biol. Chem. 1998; 273: 34616-34622Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) was performed. Coexpression of dynamin-K44A inhibited tonic internalization (Fig. 2 B), whereas GRK2-K220R and arrestin-3 (201) had no effect (data not shown). Interestingly, coexpression of dynamin-K44A, but not GRK2-K220R or arrestin-3 (201), also resulted in an ∼2-fold higher cell surface expression of TPβ as assessed by ELISA (data not shown). In contrast, dynamin-K44A had no effect on cell surface expression of TPα (data not shown). When cells were preincubated with inhibitors of clathrin-coated pit formation such as sucrose and NH4Cl, tonic internalization of TPβ was also suppressed (Fig.2 B). Whereas these results demonstrate that tonic and agonist-induced internalization of TPβ are both dynamin-dependent, tonically internalized TPβ is targeted to clathrin-coated pits via a mechanism independent of GRKs and arrestins.Because the carboxyl terminus of TPβ appears critical in tonic internalization of the receptor, we next determined whether this function could be ascribed to any particular residues. Progressive deletion mutants were first used to address this question (Fig. 1). All constructs were transiently transfected in HEK293 cells using transfection conditions that yielded comparable levels of receptor expression (∼1 pmol/mg protein). The removal of up to 63 residues from the carboxyl terminus (S344Stop) appeared to have no effect on tonic internalization (Fig. 3), whereas agonist-induced internalization was completely blocked (15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). However, truncation of an additional 7 amino acids (L337Stop) completely abolished tonic internalization (Fig. 3). Thus, the region found between residues 338 and 344 seems to play a critical role in tonic internalization of TPβ. Comparison of this region of TPβ (EYSGTIS) with the corresponding region of TPα (TQRSGLQ) suggests that Tyr-339 in TPβ might be an important component of a tonic internalization motif. A role for tyrosine-based internalization motifs in tonic endocytosis of a variety of receptors has been demonstated (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar, 6Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 22Trowbridge I.S. Collawn J.F. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar). To test this hypothesis, we generated a Y339A mutant TPβ and characterized tonic and agonist-induced internalization. Indeed, TPβ-Y339A did not undergo any tonic internalization (Fig.4), suggesting a critical role for Tyr-339 in this process. Additional amino acids between residues 338 and 344 in TPβ were then individually mutated to alanine in an attempt to identify a motif for tonic internalization. The E338A, S340A, G341A, T342A, and S344A mutants of TPβ appeared to undergo normal tonic internalization, whereas I343A was completely inhibited (Fig. 4). None of these mutations affected agonist-induced internalization of TPβ (Fig. 4 C), suggesting that the motif for tonic internalization is distinct from the region required for agonist-induced trafficking of TPβ (15Parent J.-L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Thus, our data demonstrate that the YXXXI motif plays a critical role in tonic internalization of TPβ. This sequence is closely related to the YXXφ motif identified as playing an important role in tonic internalization of the transferrin receptor, T-cell receptor (CD3), lgp-A/lamp-1, lgp-B/lamp-2, lysosomal acid phosphatase, CI-mannose-6-phosphate receptor, polymeric Ig receptor, and TGN38 receptor (23Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1331) Google Scholar).Figure 3Tonic internalization of different carboxyl tail truncation mutants of TP β. HEK293 cells were transfected with amounts of TPβ DNA yielding receptor expression of ∼1 pmol/mg protein for each construct. Cells were labeled with the FLAG antibody at 4 °C prior to being incubated at 37 °C for 1 h. A, tonic internalization of each receptor construct was analyzed by immunofluorescence. B,quantitation of tonic internalization of the different receptor constructs was performed as described in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Identification of the residues involved in tonic internalization of TP β. Amino acids between residues 338 and 344, inclusively, were individually mutated to alanines. A, immunofluorescence analysis of the tonic internalization of each receptor construct. B, quantitation of intracellular fluorescence, as described in Fig. 2. C,agonist-induced internalization of the receptor mutants. HEK293 cells were transiently transfected with FLAG epitope-tagged receptors and the percentage of receptors remaining at the cell surface after 2 h of stimulation with 100 nmU46619 was measured by ELISA analysis, as described under "Experimental Procedures." The results represent the mean ± S.E. of three independent experiments, each done in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further clarify the importance of Tyr-339 in tonic internalization, we generated Q338Y and R339Y mutants in the carboxyl terminus of TPα. Interestingly, introduction of a Tyr at position 339 in TPα, creating a YXXφ motif, induced tonic internalization of TPα (Fig.5). Mutation of Leu-342 to Ala in the R339Y mutant inhibited tonic internalization, demonstrating the importance of the hydrophobic residue in this process (Fig. 5). In an effort to determine the importance of the spacing between the Tyr and hydrophobic residues, we introduced a Thr between Gly-341 and Leu-342 in the R339Y mutant (R339Y-T342) to create a YXXXφ motif similar to that found in TPβ. This latter addition did not affect the internalization induced by the Tyr residue in R339Y, verifying that both YX 2φ and YX 3φ can function as efficient internalization motifs. Interestingly, a Q338Y mutant (also creating a YX 3φ motif but one residue closer to the plasma membrane than in TPβ and TPα (R339Y-T342)) did not induce tonic internalization of TPα. This suggests that the position of the Tyr in the receptor carboxyl tail is also an important determinant in this process. As expected, none of these mutations conferred agonist-induced internalization of TPα (data not shown). Our data suggest that both the distance between the Tyr and the hydrophobic residue and the position of the YX 2–3φ motif in the receptor carboxyl tail are important determinants in tonic internalization of the thromboxane receptor. It is interesting to note that a YLGI peptide sequence found in the second intracellular loop of both TP receptor isoforms is evidently not sufficient to induce tonic internalization because this is not observed for TPα. Moreover, mutation of residues within this motif in TPβ did not affect tonic internalization (data not shown).Figure 5Redistribution of receptors in HEK293 cells after introduction of a tyrosine residue at position 339 in the carboxyl tail of TP α. HEK293 cells were transfected with amounts of DNA yielding receptor expression of ∼1 pmol/mg of protein for each TPα construct. A,redistribution of TPα mutants was assessed by immunofluorescence.B, intracellular fluorescence was evaluated as described in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because tonic internalization has also been reported for other GPCRs (2Signoret N. Oldridge J. Pelchen-Matthews A. Klasse P.J. Tran T. Brass L.F. Rosenkilde M.M. Schwartz T.W. Holmes W. Dallas W. Luther M.A. Wells T.N.C. Hoxie J.A. Marsh M. J. Cell Biol. 1997; 139: 651-664Crossref PubMed Scopus (335) Google Scholar, 3Baratti-Elbaz C. Ghinea N. Lahuma O. Loosfelt H. Pichon Milgrom E. Mol. Endocrinol. 1999; 13: 1751-1765Crossref PubMed Scopus (70) Google Scholar, 4Roseberry A.G. Hosey M.M. J. Biol. Chem. 1999; 274: 33671-33676Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Shapiro M.J. Coughlin S.R. J. Biol. Chem. 1998; 273: 20009-29014Google Scholar), it is important to consider the biological role of this process. It has been proposed that tonic internalization of the thrombin receptor generates an intracellular pool of receptors that is used to repopulate the cell surface with functional receptors (24Shapiro M.J. Coughlin S.R. J. Biol. Chem. 1998; 273: 20009-29014Google Scholar). If a similar role can be attributed to tonic internalization of TPβ, we would expect that these receptors would recycle back to the cell surface following tonic internalization. To address this issue, cell surface receptors were labeled with M1 anti-FLAG antibody at 4 °C and then allowed to undergo tonic internalization for 1 h at 37 °C. The cells were washed briefly with PBS/EDTA to strip the cell surface antibody (which binds in a Ca2+-dependent manner), reincubated at 37 °C, fixed, and then receptor distribution determined by immunofluorescence. TPβ was only detected intracellularly after cell surface antibody was stripped with PBS/EDTA (Fig.6, panel B). However, following incubation at 37 °C, there was extensive redistribution of the intracellular receptors to the cell surface (Fig. 6, panel C). This recycling was not caused by new protein synthesis or to transport of new receptors from intracellular stores because visualized receptors originated from the initial labeling of cell surface receptors with antibody. These data suggest that there is constant recycling of the tonically internalized TPβ between the cell surface and an unidentified intracellular compartment, similar to what has been observed for the thrombin receptor (24Shapiro M.J. Coughlin S.R. J. Biol. Chem. 1998; 273: 20009-29014Google Scholar). Thus, tonic internalization of TPβ likely helps to maintain an intracellular pool of functional receptors that recycle to the cell surface to preserve agonist sensitivity.Figure 6Tonically internalized TP β receptors recycle to the cell surface. FLAG epitope-tagged receptors were transiently transfected in HEK293 cells. A,cells were labeled with the FLAG antibody at 4 °C and incubated at 37 °C for 1 h to allow tonic internalization. B, the cell surface was then stripped of the remaining FLAG antibody with two quick washes with cold PBS/EDTA (1 mm). C, cells were then reincubated for 1 h at 37 °C to permit recycling of receptors to the cell surface (see "Experimental Procedures" for details). Results shown are representative of at least five independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The YXXφ motif is one of the most extensively characterized motifs within cytosolic domains involved in the targeting of integral membrane proteins. Tyrosine-based sorting signals conforming to the YXXφ motif have been shown to interact directly with the μ1, μ2, and μ3 subunits of the adaptor complexes AP-1, AP-2, and AP-3, respectively (reviewed in Ref. 7Ohno H. Aguilar R.C. Yeh D. Taura T. Saito T. Bonifacino J.S. J. Biol. Chem. 1998; 273: 25915-25921Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The critical tyrosine does not need to be phosphorylated and, in fact, the interaction of YXXφ and μ may actually be reduced by phosphorylation (25Ohno H. Fournier M.C. Poy G. Bonifacino J.S. J. Biol. Chem. 1996; 271: 29009-29015Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 26Shiratori T. Miyatake S. Ohno H. Nkaseko C. Isono K. Bonifacino J.S. Saito T. Immunity. 1997; 6: 583-589Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The AP-1 complex associates with the trans-Golgi network and directs the transport of lysosomal enzymes to endosomes, whereas the AP-2 complex associates with the plasma membrane and directs the trafficking of cell surface proteins via clathrin-coated pits. AP-3 is involved in the delivery of proteins to lysosomes and lysosome-related organelles (27Hirst J. Bright N.A. Rous B. Robinson M.S. Mol. Biol. Cell. 1999; 10: 2787-2802Crossref PubMed Scopus (213) Google Scholar). Recent studies also suggest that there is a fourth adaptor-related protein complex, AP-4, that is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network (27Hirst J. Bright N.A. Rous B. Robinson M.S. Mol. Biol. Cell. 1999; 10: 2787-2802Crossref PubMed Scopus (213) Google Scholar, 28Dell'Angelica E.C. Mullins C. Bonifacino J.S. J. Biol. Chem. 1999; 274: 7278-7285Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The μ4 subunit of this complex specifically interacts with a tyrosine-based sorting signal, suggesting that AP-4 is also involved in the recognition and sorting of proteins with tyrosine-based motifs (27Hirst J. Bright N.A. Rous B. Robinson M.S. Mol. Biol. Cell. 1999; 10: 2787-2802Crossref PubMed Scopus (213) Google Scholar).Ohno et al. (7Ohno H. Aguilar R.C. Yeh D. Taura T. Saito T. Bonifacino J.S. J. Biol. Chem. 1998; 273: 25915-25921Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) investigated the selectivity for interaction of tyrosine-based sorting signals with μ1, μ2, μ3A, and μ3B subunits via screening of a combinatorialXXXYXXφ library using the yeast two-hybrid system. Their results revealed that there was no absolute requirement for the presence of specific residues at any of the X or φ positions. This contrasted with the critical tyrosine residue that could not be substituted by any other residue without a dramatic decrease in sorting activity (6Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 7Ohno H. Aguilar R.C. Yeh D. Taura T. Saito T. Bonifacino J.S. J. Biol. Chem. 1998; 273: 25915-25921Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 29Canfield W.M. Johnson K.F. Ye R.D. Gregory W. Kornfield S. J. Biol. Chem. 1991; 266: 5682-5688Abstract Full Te
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