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W1981653194 abstract "The β2-adrenergic receptor and δ opioid receptor represent distinct G protein-coupled receptors that undergo agonist-induced endocytosis via clathrin-coated pits but differ significantly in their postendocytic sorting between recycling and degradative membrane pathways, respectively. Previous results indicate that a distal portion of the carboxyl-terminal cytoplasmic domain of the β2-adrenergic receptor, which engages in PDZ domain-mediated protein interaction, is required for efficient recycling of receptors after agonist-induced endocytosis. Here we demonstrate that a four-residue sequence (DSLL) comprising the core of this protein interaction domain functions as a transplantable endocytic sorting signal that is sufficient to re-route endocytosed δ opioid receptor into a rapid recycling pathway, to inhibit proteolytic down-regulation of receptors, and to mediate receptor-autonomous sorting of mutant receptors from the wild type allele when co-expressed in the same cells. These observations define a transplantable signal mediating rapid recycling of a heterologous G protein-coupled receptor, and they suggest that rapid recycling of certain membrane proteins does not occur by bulk membrane flow but is instead mediated by a specific endocytic sorting mechanism. The β2-adrenergic receptor and δ opioid receptor represent distinct G protein-coupled receptors that undergo agonist-induced endocytosis via clathrin-coated pits but differ significantly in their postendocytic sorting between recycling and degradative membrane pathways, respectively. Previous results indicate that a distal portion of the carboxyl-terminal cytoplasmic domain of the β2-adrenergic receptor, which engages in PDZ domain-mediated protein interaction, is required for efficient recycling of receptors after agonist-induced endocytosis. Here we demonstrate that a four-residue sequence (DSLL) comprising the core of this protein interaction domain functions as a transplantable endocytic sorting signal that is sufficient to re-route endocytosed δ opioid receptor into a rapid recycling pathway, to inhibit proteolytic down-regulation of receptors, and to mediate receptor-autonomous sorting of mutant receptors from the wild type allele when co-expressed in the same cells. These observations define a transplantable signal mediating rapid recycling of a heterologous G protein-coupled receptor, and they suggest that rapid recycling of certain membrane proteins does not occur by bulk membrane flow but is instead mediated by a specific endocytic sorting mechanism. G protein-coupled receptor β2-adrenergic receptor δ opioid receptor hemagglutinin Dulbecco's modified Eagle's medium V2 vasopressin receptor [d-Ala2,d-Leu5 enkephalin] phosphate-buffered saline Tris-buffered saline Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa Many G protein-coupled receptors (GPCRs)1 undergo agonist-induced endocytosis via clathrin-coated pits (1von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 2Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Adv. Pharmacol. 1998; 42: 429-433Crossref PubMed Scopus (47) Google Scholar, 3Ferguson S.S. Zhang J. Barak L.S. Caron M.G. Life Sci. 1998; 62: 1561-1565Crossref PubMed Scopus (186) Google Scholar, 4Lefkowitz R.J. Pitcher J. Krueger K. Daaka Y. Adv. Pharmacol. 1998; 42: 416-420Crossref PubMed Scopus (165) Google Scholar). However, GPCRs endocytosed by this highly conserved mechanism can follow divergent downstream membrane pathways that serve distinct physiological functions (5Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). For example, both the β2-adrenergic receptor (β2AR) and δ opioid receptor (δOR) endocytose in HEK293 cells via clathrin-coated pits within several minutes after agonist-induced activation (1von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 6Zhang J. Ferguson S. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 7Chu P. Murray S. Lissin D. von Zastrow M. J. Biol. Chem. 1997; 272: 27124-27130Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Most β2ARs are recycled back to the plasma membrane within 30 min after endocytosis, whereas most internalized δORs do not recycle but instead traverse a divergent membrane pathway leading to lysosomes (8Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Rapid recycling of the β2AR is well established to play an important role in promoting functional resensitization of signal transduction (3Ferguson S.S. Zhang J. Barak L.S. Caron M.G. Life Sci. 1998; 62: 1561-1565Crossref PubMed Scopus (186) Google Scholar, 4Lefkowitz R.J. Pitcher J. Krueger K. Daaka Y. Adv. Pharmacol. 1998; 42: 416-420Crossref PubMed Scopus (165) Google Scholar), whereas trafficking of δOR to lysosomes contributes to the functionally opposite process of agonist-induced down-regulation of receptors (9Law P.-Y. Hom D.S. Loh H.H. J. Biol. Chem. 1984; 259: 4096-4104Abstract Full Text PDF PubMed Google Scholar, 10Law P.Y. Loh H.H. J. Pharm. Exp. Ther. 1999; 289: 607-624PubMed Google Scholar, 11Tsao P. Cao T. von Zastrow M. Trends Pharmacol. Sci. 2001; 22: 91-96Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Recent studies have identified additional functions of endocytosis in mediating signal transduction and suggest the existence of additional complexity in the post-endocytic membrane trafficking of certain GPCRs (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 13Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 15Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar, 16Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar). However, little is known about mechanisms that determine the specificity with which GPCRs are sorted between distinct membrane pathways after endocytosis.In general it is thought that cytoplasmic domains of membrane proteins contain structural elements that function as sorting “signals” to control specific steps of intracellular trafficking (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 18Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (550) Google Scholar). Previous studies indicate that the carboxyl-terminal cytoplasmic domain of certain GPCRs contains sequences that promote receptor endocytic trafficking to lysosomes (19Trapaidze N. Keith D.E. Cvejic S. Evans C.J. Devi L.A. J. Biol. Chem. 1996; 271: 29279-29285Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Cvejic S. Trapaidze N. Cyr C. Devi L.A. J. Biol. Chem. 1996; 271: 4073-4076Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In contrast, recycling of internalized membrane proteins back to the plasma membrane is generally thought to occur by “default” without any requirement for cytoplasmic sorting signals (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 18Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (550) Google Scholar). Support for this hypothesis includes previous studies establishing that major lipid constituents of the plasma membrane recycle rapidly by “bulk flow” (22Hao M. Maxfield F.R. J. Biol. Chem. 2000; 275: 15279-15286Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) and that certain integral membrane proteins recycle rapidly in the absence of any exposed cytoplasmic residues (23Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (413) Google Scholar).Emerging evidence suggests that recycling of certain GPCRs may not occur by default but may require specific membrane sorting signals. Endocytosed V2 vasopressin receptors (V2Rs) recycle to the plasma membrane by a membrane pathway characterized by its remarkably slow kinetics (t12 > 2 h) (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar). Recycling of receptors via this “long pathway” requires a specific sequence present in the cytoplasmic tail of the V2R (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 24Innamorati G. Sadeghi H. Birnbaumer M. J. Recept. Signal Transduct. Res. 1999; 19: 315-326Crossref PubMed Scopus (30) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar), and this sequence is sufficient to act as a sorting signal to cause a chimeric mutant V1 vasopressin receptor (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) or β2AR (25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar) to traverse this distinct recycling pathway. However, disruption of this sorting signal in the V2R causes internalized receptors to recycle with similarly rapid kinetics (t12 < 30 min) as the wild type β2AR (25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Although these observations confirm that recycling of GPCRs by the specialized long pathway is mediated by a specific cytoplasmic sorting signal, they also support the hypothesis that more rapid recycling of GPCRs occurs by default.A previous study of the β2AR suggested that rapid recycling of certain GPCRs may require a specific sorting signal. Mutations of a sequence present in the distal portion of the carboxyl-terminal cytoplasmic domain of the β2AR, which disrupt a specific interaction with the NHERF (Na+/H+ exchanger regulatory factor)/EBP50 (ezrin/radixin/moesin-binding phosphoprotein of 50 kDa) family of PDZ domain-containing proteins (27Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar, 28Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (310) Google Scholar, 29Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar), strongly inhibited recycling of receptors after agonist-induced endocytosis (30Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (562) Google Scholar). However, as NHERF/EBP50 proteins play multiple important roles in cell physiology (including controlling ion transport across the plasma membrane (Refs.27Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar and 31Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar), contributing to the structure of the cortical actin cytoskeleton (Refs. 29Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar and 32Pearson M.A. Reczek D. Bretscher A. Karplus P.A. Cell. 2000; 101: 259-270Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar), and cross-linking certain proteins in the plasma membrane (Ref. 31Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar)), impaired recycling of tail mutant β2ARs may not indicate the existence of a specific recycling signal but might instead reflect a secondary consequence of disrupting another aspect of receptor function or membrane organization. Furthermore, as the specific sequence required for high affinity interaction of the β2AR with NHERF/EBP50 family proteins is not conserved in most other GPCRs (33Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar), it was not established whether this PDZ-interacting sequence could play any role in controlling the membrane trafficking of a distinct GPCR.We have addressed these questions by examining whether sequences derived from the carboxyl-terminal cytoplasmic domain of the β2AR are sufficient to function as a transplantable sorting signal to promote rapid recycling of a heterologous GPCR. We have focused on studying effects on the endocytic trafficking of an epitope-tagged version of δOR expressed in HEK293 cells, where this GPCR is well established to endocytose via clathrin-coated pits but differs substantially in its postendocytic sorting from the β2AR even when co-expressed at similar levels in the same cells (8Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Our results indicate that the distal tail sequence from the β2AR can indeed function as an autonomous sorting signal, which is fully sufficient to re-route endocytosed δOR into a rapid recycling pathway. This transplantable sorting activity is functionally significant because it also confers reduced proteolytic down-regulation on mutant receptors, and it is possible to reduce the sorting signal sufficient to mediate both effects to a four-residue sequence (DSLL) corresponding to the minimal structure required to mediate detectable binding of the mutant receptor tail to NHERF/EBP50 family proteins. The autonomous activity of this sorting signal is demonstrated by the ability of the four-residue sequence to selectively re-route trafficking of a mutant δOR without causing any detectable effect on the endocytic trafficking of the co-expressed wild type allele. Thus, at least in the case of certain GPCRs, rapid recycling does not occur by default but can instead be mediated by a specific signal-dependent sorting operation.DISCUSSIONIn this study we examined the effect of transplanting sequences derived from the carboxyl-terminal cytoplasmic domain of the β2AR on the endocytic trafficking of the δOR. These experiments were motivated by the fact that, although both the β2AR and δOR are co-endocytosed in HEK293 by clathrin-coated pits, these distinct GPCRs differ substantially in their postendocytic trafficking between recycling and degradative membrane pathways, respectively. Furthermore, as these GPCRs signal via coupling to distinct heterotrimeric G proteins, the sorting activity of the β2AR-derived sorting signal does not appear to be limited to Gs-coupled GPCRs. A previous study indicated that a distal portion of the carboxyl-terminal cytoplasmic domain is necessary for rapid recycling of the β2AR. However, as the NHERF/EBP50 family proteins that interact with this sequence serve multiple functions in cell physiology and the sequence required for this protein interaction is not conserved in most other GPCRs (including δOR), it was not determined whether the requirement of this sequence for recycling of receptors reflects its activity as an “autonomous” sorting signal or an indirect consequence perturbing other aspects of cellular function or β2AR signaling activity. The present results demonstrate that this PDZ domain-binding sequence is sufficient to re-route the endocytic trafficking of δOR from a lysosomal degradative pathway into a rapid recycling pathway and to mediate autonomous sorting of a mutant δOR from the wild type δOR when co-expressed in the same cells. Thus, we conclude that the β2AR does indeed contain an autonomous, transplantable endocytic sorting signal, which is sufficient to re-route a heterologous GPCR into a rapid recycling pathway and to cause functionally significant changes in agonist-induced down-regulation of receptors. To our knowledge, this is the first direct demonstration of the existence of a modular sorting signal mediating rapid recycling of any GPCR.It has been well documented that GPCRs contain cytoplasmic sequences that influence specific membrane trafficking steps. For example, the D1 dopamine receptor contains a sequence in the carboxyl-terminal cytoplasmic domain that influences trafficking of newly synthesized receptors in the biosynthetic pathway (46Bermak J.C. Li M. Bullock C. Zhou Q.Y. Na. Cell Biol. 2001; 3: 492-498Crossref PubMed Scopus (229) Google Scholar). Studies of the PAR1 thrombin receptor and thromboxane A2 receptor have identified distinct portions of the carboxyl-terminal cytoplasmic domain that mediate regulated and constitutive endocytosis (47Shapiro 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, 48Parent 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). Studies of PAR1 have also identified a distinct function of the carboxyl-terminal cytoplasmic domain in promoting receptor trafficking to lysosomes (21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Recent studies of the V2 vasopressin receptor identified a cytoplasmic sequence that mediates trafficking of receptors via a specialized recycling pathway characterized by its remarkably slow kinetics (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, as disruption of this sequence causes rapid recycling of receptors (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), these studies suggest that only slow pathway(s) of GPCR recycling are mediated by specific sorting signals and more rapid recycling can occur by default. The present results demonstrate the existence of a modular sorting signal that specifically promotes rapid recycling of a heterologous GPCR.The idea that rapid recycling can occur by default is supported by a large number of previous studies of various membrane proteins (49Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar). For example, elegant studies of endocytic trafficking of the transferrin receptor indicate that rapid recycling of this transmembrane protein can occur after removal of all exposed cytoplasmic residues (23Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (413) Google Scholar). Similar experiments suggest that the epidermal growth factor receptor can recycle to the plasma membrane in the absence of any specific sequence present in the cytoplasmic domain of the receptor (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar). Moreover, as discussed above, truncation and point mutations of the portion of the V2R tail that abolish slow recycling via the long pathway cause receptors to recycle to the plasma membrane with rapid kinetics (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These considerations suggest that the rapid recycling pathway mediated by the β2AR-derived tail sequence represents a specialized mechanism of receptor regulation, consistent with previous observations suggesting that the mechanisms mediating rapid recycling of the β2AR and transferrin receptors are distinguishable (30Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (562) Google Scholar). In addition, they expand on previous evidence suggesting that specialized membrane trafficking mechanisms may play an important role in distinguishing the functional regulation of specific GPCRs (5Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 16Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 50Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Crossref PubMed Scopus (117) Google Scholar). Although our studies have focused exclusively on the functional role of endocytic sorting in controlling proteolytic down-regulation mediated by receptor trafficking to lysosomes, we note that multiple mechanisms can contribute to down-regulation of GPCRs under physiological conditions (51Tsao P. von Zastrow M. Curr. Opin. Neurobiol. 2000; 10: 365-369Crossref PubMed Scopus (133) Google Scholar). In particular, recent studies provide strong evidence for an important role of proteasomes in mediating proteolysis of opioid receptors (52Petaja-Repo U.E. Hogue M. Laperriere A. Bhalla S. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,53Chaturvedi K. Bandari P. Chinen N. Howells R.D. J. Biol. Chem. 2001; 276: 12345-12355Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and additional mechanism(s) contributing to proteolysis of other GPCRs (54Kojro E. Fahrenholz F. J. Biol. Chem. 1995; 270: 6476-6481Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 55Jockers R. Angers S. Da Silva A. Benaroch P. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1999; 274: 28900-28908Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In future studies it will be interesting to determine what physiological significance this remarkable diversity of mechanisms mediating both recycling and proteolysis of GPCRs might have. Many G protein-coupled receptors (GPCRs)1 undergo agonist-induced endocytosis via clathrin-coated pits (1von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 2Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Adv. Pharmacol. 1998; 42: 429-433Crossref PubMed Scopus (47) Google Scholar, 3Ferguson S.S. Zhang J. Barak L.S. Caron M.G. Life Sci. 1998; 62: 1561-1565Crossref PubMed Scopus (186) Google Scholar, 4Lefkowitz R.J. Pitcher J. Krueger K. Daaka Y. Adv. Pharmacol. 1998; 42: 416-420Crossref PubMed Scopus (165) Google Scholar). However, GPCRs endocytosed by this highly conserved mechanism can follow divergent downstream membrane pathways that serve distinct physiological functions (5Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). For example, both the β2-adrenergic receptor (β2AR) and δ opioid receptor (δOR) endocytose in HEK293 cells via clathrin-coated pits within several minutes after agonist-induced activation (1von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 6Zhang J. Ferguson S. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 7Chu P. Murray S. Lissin D. von Zastrow M. J. Biol. Chem. 1997; 272: 27124-27130Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Most β2ARs are recycled back to the plasma membrane within 30 min after endocytosis, whereas most internalized δORs do not recycle but instead traverse a divergent membrane pathway leading to lysosomes (8Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Rapid recycling of the β2AR is well established to play an important role in promoting functional resensitization of signal transduction (3Ferguson S.S. Zhang J. Barak L.S. Caron M.G. Life Sci. 1998; 62: 1561-1565Crossref PubMed Scopus (186) Google Scholar, 4Lefkowitz R.J. Pitcher J. Krueger K. Daaka Y. Adv. Pharmacol. 1998; 42: 416-420Crossref PubMed Scopus (165) Google Scholar), whereas trafficking of δOR to lysosomes contributes to the functionally opposite process of agonist-induced down-regulation of receptors (9Law P.-Y. Hom D.S. Loh H.H. J. Biol. Chem. 1984; 259: 4096-4104Abstract Full Text PDF PubMed Google Scholar, 10Law P.Y. Loh H.H. J. Pharm. Exp. Ther. 1999; 289: 607-624PubMed Google Scholar, 11Tsao P. Cao T. von Zastrow M. Trends Pharmacol. Sci. 2001; 22: 91-96Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Recent studies have identified additional functions of endocytosis in mediating signal transduction and suggest the existence of additional complexity in the post-endocytic membrane trafficking of certain GPCRs (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 13Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 15Claing A. Perry S.J. Achiriloaie M. Walker J.K. Albanesi J.P. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1119-1124Crossref PubMed Scopus (144) Google Scholar, 16Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar). However, little is known about mechanisms that determine the specificity with which GPCRs are sorted between distinct membrane pathways after endocytosis. In general it is thought that cytoplasmic domains of membrane proteins contain structural elements that function as sorting “signals” to control specific steps of intracellular trafficking (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 18Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (550) Google Scholar). Previous studies indicate that the carboxyl-terminal cytoplasmic domain of certain GPCRs contains sequences that promote receptor endocytic trafficking to lysosomes (19Trapaidze N. Keith D.E. Cvejic S. Evans C.J. Devi L.A. J. Biol. Chem. 1996; 271: 29279-29285Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Cvejic S. Trapaidze N. Cyr C. Devi L.A. J. Biol. Chem. 1996; 271: 4073-4076Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In contrast, recycling of internalized membrane proteins back to the plasma membrane is generally thought to occur by “default” without any requirement for cytoplasmic sorting signals (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar, 18Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (550) Google Scholar). Support for this hypothesis includes previous studies establishing that major lipid constituents of the plasma membrane recycle rapidly by “bulk flow” (22Hao M. Maxfield F.R. J. Biol. Chem. 2000; 275: 15279-15286Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) and that certain integral membrane proteins recycle rapidly in the absence of any exposed cytoplasmic residues (23Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (413) Google Scholar). Emerging evidence suggests that recycling of certain GPCRs may not occur by default but may require specific membrane sorting signals. Endocytosed V2 vasopressin receptors (V2Rs) recycle to the plasma membrane by a membrane pathway characterized by its remarkably slow kinetics (t12 > 2 h) (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar). Recycling of receptors via this “long pathway” requires a specific sequence present in the cytoplasmic tail of the V2R (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 24Innamorati G. Sadeghi H. Birnbaumer M. J. Recept. Signal Transduct. Res. 1999; 19: 315-326Crossref PubMed Scopus (30) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar), and this sequence is sufficient to act as a sorting signal to cause a chimeric mutant V1 vasopressin receptor (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) or β2AR (25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar) to traverse this distinct recycling pathway. However, disruption of this sorting signal in the V2R causes internalized receptors to recycle with similarly rapid kinetics (t12 < 30 min) as the wild type β2AR (25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). Although these observations confirm that recycling of GPCRs by the specialized long pathway is mediated by a specific cytoplasmic sorting signal, they also support the hypothesis that more rapid recycling of GPCRs occurs by default. A previous study of the β2AR suggested that rapid recycling of certain GPCRs may require a specific sorting signal. Mutations of a sequence present in the distal portion of the carboxyl-terminal cytoplasmic domain of the β2AR, which disrupt a specific interaction with the NHERF (Na+/H+ exchanger regulatory factor)/EBP50 (ezrin/radixin/moesin-binding phosphoprotein of 50 kDa) family of PDZ domain-containing proteins (27Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar, 28Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (310) Google Scholar, 29Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar), strongly inhibited recycling of receptors after agonist-induced endocytosis (30Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (562) Google Scholar). However, as NHERF/EBP50 proteins play multiple important roles in cell physiology (including controlling ion transport across the plasma membrane (Refs.27Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar and 31Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar), contributing to the structure of the cortical actin cytoskeleton (Refs. 29Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar and 32Pearson M.A. Reczek D. Bretscher A. Karplus P.A. Cell. 2000; 101: 259-270Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar), and cross-linking certain proteins in the plasma membrane (Ref. 31Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar)), impaired recycling of tail mutant β2ARs may not indicate the existence of a specific recycling signal but might instead reflect a secondary consequence of disrupting another aspect of receptor function or membrane organization. Furthermore, as the specific sequence required for high affinity interaction of the β2AR with NHERF/EBP50 family proteins is not conserved in most other GPCRs (33Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar), it was not established whether this PDZ-interacting sequence could play any role in controlling the membrane trafficking of a distinct GPCR. We have addressed these questions by examining whether sequences derived from the carboxyl-terminal cytoplasmic domain of the β2AR are sufficient to function as a transplantable sorting signal to promote rapid recycling of a heterologous GPCR. We have focused on studying effects on the endocytic trafficking of an epitope-tagged version of δOR expressed in HEK293 cells, where this GPCR is well established to endocytose via clathrin-coated pits but differs substantially in its postendocytic sorting from the β2AR even when co-expressed at similar levels in the same cells (8Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Our results indicate that the distal tail sequence from the β2AR can indeed function as an autonomous sorting signal, which is fully sufficient to re-route endocytosed δOR into a rapid recycling pathway. This transplantable sorting activity is functionally significant because it also confers reduced proteolytic down-regulation on mutant receptors, and it is possible to reduce the sorting signal sufficient to mediate both effects to a four-residue sequence (DSLL) corresponding to the minimal structure required to mediate detectable binding of the mutant receptor tail to NHERF/EBP50 family proteins. The autonomous activity of this sorting signal is demonstrated by the ability of the four-residue sequence to selectively re-route trafficking of a mutant δOR without causing any detectable effect on the endocytic trafficking of the co-expressed wild type allele. Thus, at least in the case of certain GPCRs, rapid recycling does not occur by default but can instead be mediated by a specific signal-dependent sorting operation. DISCUSSIONIn this study we examined the effect of transplanting sequences derived from the carboxyl-terminal cytoplasmic domain of the β2AR on the endocytic trafficking of the δOR. These experiments were motivated by the fact that, although both the β2AR and δOR are co-endocytosed in HEK293 by clathrin-coated pits, these distinct GPCRs differ substantially in their postendocytic trafficking between recycling and degradative membrane pathways, respectively. Furthermore, as these GPCRs signal via coupling to distinct heterotrimeric G proteins, the sorting activity of the β2AR-derived sorting signal does not appear to be limited to Gs-coupled GPCRs. A previous study indicated that a distal portion of the carboxyl-terminal cytoplasmic domain is necessary for rapid recycling of the β2AR. However, as the NHERF/EBP50 family proteins that interact with this sequence serve multiple functions in cell physiology and the sequence required for this protein interaction is not conserved in most other GPCRs (including δOR), it was not determined whether the requirement of this sequence for recycling of receptors reflects its activity as an “autonomous” sorting signal or an indirect consequence perturbing other aspects of cellular function or β2AR signaling activity. The present results demonstrate that this PDZ domain-binding sequence is sufficient to re-route the endocytic trafficking of δOR from a lysosomal degradative pathway into a rapid recycling pathway and to mediate autonomous sorting of a mutant δOR from the wild type δOR when co-expressed in the same cells. Thus, we conclude that the β2AR does indeed contain an autonomous, transplantable endocytic sorting signal, which is sufficient to re-route a heterologous GPCR into a rapid recycling pathway and to cause functionally significant changes in agonist-induced down-regulation of receptors. To our knowledge, this is the first direct demonstration of the existence of a modular sorting signal mediating rapid recycling of any GPCR.It has been well documented that GPCRs contain cytoplasmic sequences that influence specific membrane trafficking steps. For example, the D1 dopamine receptor contains a sequence in the carboxyl-terminal cytoplasmic domain that influences trafficking of newly synthesized receptors in the biosynthetic pathway (46Bermak J.C. Li M. Bullock C. Zhou Q.Y. Na. Cell Biol. 2001; 3: 492-498Crossref PubMed Scopus (229) Google Scholar). Studies of the PAR1 thrombin receptor and thromboxane A2 receptor have identified distinct portions of the carboxyl-terminal cytoplasmic domain that mediate regulated and constitutive endocytosis (47Shapiro 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, 48Parent 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). Studies of PAR1 have also identified a distinct function of the carboxyl-terminal cytoplasmic domain in promoting receptor trafficking to lysosomes (21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Recent studies of the V2 vasopressin receptor identified a cytoplasmic sequence that mediates trafficking of receptors via a specialized recycling pathway characterized by its remarkably slow kinetics (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, as disruption of this sequence causes rapid recycling of receptors (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), these studies suggest that only slow pathway(s) of GPCR recycling are mediated by specific sorting signals and more rapid recycling can occur by default. The present results demonstrate the existence of a modular sorting signal that specifically promotes rapid recycling of a heterologous GPCR.The idea that rapid recycling can occur by default is supported by a large number of previous studies of various membrane proteins (49Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar). For example, elegant studies of endocytic trafficking of the transferrin receptor indicate that rapid recycling of this transmembrane protein can occur after removal of all exposed cytoplasmic residues (23Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (413) Google Scholar). Similar experiments suggest that the epidermal growth factor receptor can recycle to the plasma membrane in the absence of any specific sequence present in the cytoplasmic domain of the receptor (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar). Moreover, as discussed above, truncation and point mutations of the portion of the V2R tail that abolish slow recycling via the long pathway cause receptors to recycle to the plasma membrane with rapid kinetics (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These considerations suggest that the rapid recycling pathway mediated by the β2AR-derived tail sequence represents a specialized mechanism of receptor regulation, consistent with previous observations suggesting that the mechanisms mediating rapid recycling of the β2AR and transferrin receptors are distinguishable (30Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (562) Google Scholar). In addition, they expand on previous evidence suggesting that specialized membrane trafficking mechanisms may play an important role in distinguishing the functional regulation of specific GPCRs (5Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 16Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 50Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Crossref PubMed Scopus (117) Google Scholar). Although our studies have focused exclusively on the functional role of endocytic sorting in controlling proteolytic down-regulation mediated by receptor trafficking to lysosomes, we note that multiple mechanisms can contribute to down-regulation of GPCRs under physiological conditions (51Tsao P. von Zastrow M. Curr. Opin. Neurobiol. 2000; 10: 365-369Crossref PubMed Scopus (133) Google Scholar). In particular, recent studies provide strong evidence for an important role of proteasomes in mediating proteolysis of opioid receptors (52Petaja-Repo U.E. Hogue M. Laperriere A. Bhalla S. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,53Chaturvedi K. Bandari P. Chinen N. Howells R.D. J. Biol. Chem. 2001; 276: 12345-12355Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and additional mechanism(s) contributing to proteolysis of other GPCRs (54Kojro E. Fahrenholz F. J. Biol. Chem. 1995; 270: 6476-6481Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 55Jockers R. Angers S. Da Silva A. Benaroch P. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1999; 274: 28900-28908Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In future studies it will be interesting to determine what physiological significance this remarkable diversity of mechanisms mediating both recycling and proteolysis of GPCRs might have. In this study we examined the effect of transplanting sequences derived from the carboxyl-terminal cytoplasmic domain of the β2AR on the endocytic trafficking of the δOR. These experiments were motivated by the fact that, although both the β2AR and δOR are co-endocytosed in HEK293 by clathrin-coated pits, these distinct GPCRs differ substantially in their postendocytic trafficking between recycling and degradative membrane pathways, respectively. Furthermore, as these GPCRs signal via coupling to distinct heterotrimeric G proteins, the sorting activity of the β2AR-derived sorting signal does not appear to be limited to Gs-coupled GPCRs. A previous study indicated that a distal portion of the carboxyl-terminal cytoplasmic domain is necessary for rapid recycling of the β2AR. However, as the NHERF/EBP50 family proteins that interact with this sequence serve multiple functions in cell physiology and the sequence required for this protein interaction is not conserved in most other GPCRs (including δOR), it was not determined whether the requirement of this sequence for recycling of receptors reflects its activity as an “autonomous” sorting signal or an indirect consequence perturbing other aspects of cellular function or β2AR signaling activity. The present results demonstrate that this PDZ domain-binding sequence is sufficient to re-route the endocytic trafficking of δOR from a lysosomal degradative pathway into a rapid recycling pathway and to mediate autonomous sorting of a mutant δOR from the wild type δOR when co-expressed in the same cells. Thus, we conclude that the β2AR does indeed contain an autonomous, transplantable endocytic sorting signal, which is sufficient to re-route a heterologous GPCR into a rapid recycling pathway and to cause functionally significant changes in agonist-induced down-regulation of receptors. To our knowledge, this is the first direct demonstration of the existence of a modular sorting signal mediating rapid recycling of any GPCR. It has been well documented that GPCRs contain cytoplasmic sequences that influence specific membrane trafficking steps. For example, the D1 dopamine receptor contains a sequence in the carboxyl-terminal cytoplasmic domain that influences trafficking of newly synthesized receptors in the biosynthetic pathway (46Bermak J.C. Li M. Bullock C. Zhou Q.Y. Na. Cell Biol. 2001; 3: 492-498Crossref PubMed Scopus (229) Google Scholar). Studies of the PAR1 thrombin receptor and thromboxane A2 receptor have identified distinct portions of the carboxyl-terminal cytoplasmic domain that mediate regulated and constitutive endocytosis (47Shapiro 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, 48Parent 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). Studies of PAR1 have also identified a distinct function of the carboxyl-terminal cytoplasmic domain in promoting receptor trafficking to lysosomes (21Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Recent studies of the V2 vasopressin receptor identified a cytoplasmic sequence that mediates trafficking of receptors via a specialized recycling pathway characterized by its remarkably slow kinetics (12Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (110) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, as disruption of this sequence causes rapid recycling of receptors (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), these studies suggest that only slow pathway(s) of GPCR recycling are mediated by specific sorting signals and more rapid recycling can occur by default. The present results demonstrate the existence of a modular sorting signal that specifically promotes rapid recycling of a heterologous GPCR. The idea that rapid recycling can occur by default is supported by a large number of previous studies of various membrane proteins (49Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar). For example, elegant studies of endocytic trafficking of the transferrin receptor indicate that rapid recycling of this transmembrane protein can occur after removal of all exposed cytoplasmic residues (23Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (413) Google Scholar). Similar experiments suggest that the epidermal growth factor receptor can recycle to the plasma membrane in the absence of any specific sequence present in the cytoplasmic domain of the receptor (17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar). Moreover, as discussed above, truncation and point mutations of the portion of the V2R tail that abolish slow recycling via the long pathway cause receptors to recycle to the plasma membrane with rapid kinetics (26Innamorati G. Le Gouill C. Balamotis M. Birnbaumer M. J. Biol. Chem. 2001; 276: 13096-13103Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These considerations suggest that the rapid recycling pathway mediated by the β2AR-derived tail sequence represents a specialized mechanism of receptor regulation, consistent with previous observations suggesting that the mechanisms mediating rapid recycling of the β2AR and transferrin receptors are distinguishable (30Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (562) Google Scholar). In addition, they expand on previous evidence suggesting that specialized membrane trafficking mechanisms may play an important role in distinguishing the functional regulation of specific GPCRs (5Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (678) Google Scholar, 16Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar, 25Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 50Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Crossref PubMed Scopus (117) Google Scholar). Although our studies have focused exclusively on the functional role of endocytic sorting in controlling proteolytic down-regulation mediated by receptor trafficking to lysosomes, we note that multiple mechanisms can contribute to down-regulation of GPCRs under physiological conditions (51Tsao P. von Zastrow M. Curr. Opin. Neurobiol. 2000; 10: 365-369Crossref PubMed Scopus (133) Google Scholar). In particular, recent studies provide strong evidence for an important role of proteasomes in mediating proteolysis of opioid receptors (52Petaja-Repo U.E. Hogue M. Laperriere A. Bhalla S. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,53Chaturvedi K. Bandari P. Chinen N. Howells R.D. J. Biol. Chem. 2001; 276: 12345-12355Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and additional mechanism(s) contributing to proteolysis of other GPCRs (54Kojro E. Fahrenholz F. J. Biol. Chem. 1995; 270: 6476-6481Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 55Jockers R. Angers S. Da Silva A. Benaroch P. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1999; 274: 28900-28908Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In future studies it will be interesting to determine what physiological significance this remarkable diversity of mechanisms mediating both recycling and proteolysis of GPCRs might have. We thank Heather Deacon, Denise Kenski, Aaron Marley, and Michael Tanowitz for valuable advice and assistance." @default.
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W1981653194 title "A Transplantable Sorting Signal That Is Sufficient to Mediate Rapid Recycling of G Protein-coupled Receptors" @default.
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