Interactions with PDZ Domain Proteins PIST/GOPC and PDZK1 Regulate Intracellular Sorting of the Somatostatin Receptor Subtype 5

By yeast two-hybrid screening we have identified interaction partners for the intracellular C-terminal tail of the human and rodent somatostatin receptor subtype 5 (SSTR5). Interactions with the PDZ domain-containing proteins PIST and PDZK1 are mediated by the PDZ ligand motif at the C terminus of the receptor; in case of the human and mouse (but not the rat) receptors, a slight sequence variation of this motif also allows for binding of the peroxisomal receptor PEX5. PIST is Golgi-associated and retains SSTR5 in the Golgi apparatus when coexpressed with the receptor; PDZK1 on the other hand associates with the SSTR5 at the plasma membrane. Endogenous SSTR5 in the neuroendocrine AtT-20 tumor cell line is colocalized with PIST in the Golgi apparatus. On a functional level, removal of the PDZ ligand motif of the receptor does not interfere with agonist-dependent internalization of the receptor or its targeting to a Golgi-associated compartment; however, recycling of the receptor to the plasma membrane after washout of the agonist is inhibited, suggesting that the PDZ-mediated interaction of SSTR5 is required for postendocytic sorting. By yeast two-hybrid screening we have identified interaction partners for the intracellular C-terminal tail of the human and rodent somatostatin receptor subtype 5 (SSTR5). Interactions with the PDZ domain-containing proteins PIST and PDZK1 are mediated by the PDZ ligand motif at the C terminus of the receptor; in case of the human and mouse (but not the rat) receptors, a slight sequence variation of this motif also allows for binding of the peroxisomal receptor PEX5. PIST is Golgi-associated and retains SSTR5 in the Golgi apparatus when coexpressed with the receptor; PDZK1 on the other hand associates with the SSTR5 at the plasma membrane. Endogenous SSTR5 in the neuroendocrine AtT-20 tumor cell line is colocalized with PIST in the Golgi apparatus. On a functional level, removal of the PDZ ligand motif of the receptor does not interfere with agonist-dependent internalization of the receptor or its targeting to a Golgi-associated compartment; however, recycling of the receptor to the plasma membrane after washout of the agonist is inhibited, suggesting that the PDZ-mediated interaction of SSTR5 is required for postendocytic sorting. In recent years it has been appreciated that the signaling and the subcellular distribution of G-protein-coupled receptors may be influenced by proteins that interact with the intracellular regions of the receptors, most notably the C termini (1Kreienkamp H.J. Curr. Opin. Pharmacol. 2002; 2: 581-586Crossref PubMed Scopus (53) Google Scholar, 2Bockaert J. Fagni L. Dumuis A. Marin P. Pharmacol. Ther. 2004; 103: 203-221Crossref PubMed Scopus (222) Google Scholar). Several interactions have been mapped to the membrane proximal domain of the C terminus, including the so-called helix 8, which encompasses the palmitoylation motifs found in most receptors of the rhodopsin-related type I family (3Whistler J.L. Enquist J. Marley A. Fong J. Gladher F. Tsuruda P. Murray S.R. Von Zastrow M. Science. 2002; 297: 615-620Crossref PubMed Scopus (275) Google Scholar, 4Simonin F. Karcher P. Boeuf J.J. Matifas A. Kieffer B.L. J. Neurochem. 2004; 89: 766-775Crossref PubMed Scopus (85) Google Scholar, 5Heydorn A. Sondergaard B.P. Ersboll B. Holst B. Nielsen F.C. Haft C.R. Whistler J. Schwartz T.W. J. Biol. Chem. 2004; 279: 54291-54303Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). These contacts especially involve proteins that act in the postendocytic, intracellular sorting of multiple receptors, i.e. the sorting nexin SNX1, N-ethylmaleimide sensitive factor and the G-protein-coupled receptor-associated sorting protein GASP. A second type of interactions involves the distal C termini of GPCRs, 4The abbreviations used are: GPCR, G-protein-coupled receptor; CFTR; cystic fibrosis transmembrane regulator; GST, glutathione S-transferase; HEK, human embryonic kidney; PEX5, Peroxin 5. PDZ, PSD-95/discs large/zonula occludens 1; PDZK1, PDZ protein expressed in kidney 1; PIST, PDZ protein interacting specifically with Tc10; PSD-95, postsynaptic density protein of 95 kDa; SST, somatostatin; SSTR, somatostatin receptor; TGN, trans-Golgi network; EGFP, enhanced green fluorescent protein. which in many cases contain motifs for the interaction with PDZ domains. PDZ domain proteins frequently act as scaffold molecules because of their multiple protein interaction motifs (6Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1043) Google Scholar). Thus this type of interaction has the potential to target a receptor to specific subcellular domains and into specific signaling complexes. Interaction of the β1-adrenergic receptor with the third PDZ domain of PSD-95, for example, is believed to anchor this receptor at the postsynaptic density of excitatory synapses (7Hu L.A. Tang Y. Miller W.E. Cong M. Lau A.G. Lefkowitz R.J. Hall R.A. J. Biol. Chem. 2000; 275: 38659-38666Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The interaction of the parathyroid hormone receptor with Na+/H+ exchanger regulatory factor/EBP-50 on the other hand physically links the receptor to phospholipase Cβ, thereby shifting the second messenger response from adenylate cyclase activation to the hydrolysis of phospholipids (8Mahon M.J. Donowitz M. Yun C.C. Segre G.V. Nature. 2002; 417: 858-861Crossref PubMed Scopus (273) Google Scholar). We have recently initiated a search for intracellular interaction partners for the various members of the somatostatin receptor (SSTR) family (9Zitzer H. Hönck H.H. Bächner D. Richter D. Kreienkamp H.J. J. Biol. Chem. 1999; 274: 32997-33001Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 10Schwärzler A. Kreienkamp H.J. Richter D. J. Biol. Chem. 2000; 275: 9557-9562Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). There are five SSTR subtypes in mammalian species, which form a rather homogeneous subfamily within the larger family of GPCRs, as they all preferentially couple to inhibitory, pertussis toxin-sensitive G-proteins and exhibit common intracellular coupling patterns such as inhibition of adenylate cyclase (11Patel Y.C. Greenwood M.T. Warszynska A. Panetta R. Srikant C.B. Biochem. Biophys. Res. Commun. 1994; 198: 605-612Crossref PubMed Scopus (218) Google Scholar) and activation of GIRK potassium channels (12Kreienkamp H.J. Hönck H.H. Richter D. FEBS Lett. 1997; 419: 92-94Crossref PubMed Scopus (70) Google Scholar). Thus, SSTR1 was detected on the axon terminals of hypothalamic neurons extending to the median eminence (13Helboe L. Stidsen C.E. Moller M. J. Neurosci. 1998; 18: 4938-4945Crossref PubMed Google Scholar), SSTR4 was found on dendrites in hippocampal neurons (14Schreff M. Schulz S. Handel M. Keilhoff G. Braun H. Pereira G. Klutzny M. Schmidt H. Wolf G. Höllt V. J. Neurosci. 2000; 20: 3785-3797Crossref PubMed Google Scholar), and SSTR3 is localized to neuronal cilia in the rodent brain (15Handel M. Schulz S. Stanarius A. Schreff M. Erdtmann-Vourliotis M. Schmidt H. Wolf G. Höllt V. Neuroscience. 1999; 89: 909-926Crossref PubMed Scopus (308) Google Scholar). The SSTR5 is of particular pharmacological relevance as this receptor subtype regulates growth hormone secretion from the pituitary as well as insulin secretion from pancreatic β-cells (16Bruns C. Weckbecker G. Raulf F. Lübbert H. Hoyer D. CIBA Found Symp. 1995; 190 (Discussion 101-110): 89-101PubMed Google Scholar, 17Strowski M.Z. Parmar R.M. Blake A.D. Schaeffer J.M. Endocrinology. 2000; 141: 111-117Crossref PubMed Scopus (183) Google Scholar). In the AtT20 cell line, a widely used model for the regulation of hormone release by somatostatin, SSTR2 is present at the cell surface whereas SSTR5 is largely confined to the trans-Golgi-network (TGN, Ref. 18Sarret P. Esdaile M.J. McPherson P.S. Schonbrunn A. Kreienkamp H.J. Beaudet A. J. Biol. Chem. 2004; 279: 8029-8037Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Here we identify the PDZ domain proteins PIST and PDZK1 as intracellular binding partners of the SSTR5. Our data indicate that these proteins determine the intracellular sorting of the receptor; in particular, the PDZ-mediated interactions of SSTR5 appear to regulate targeting of the receptor from the Golgi/TGN to the plasma membrane. Yeast Two-hybrid Screen—A cDNA fragment coding for the C-terminal, intracellular domain of the human SSTR5 (amino acids 324–364) was amplified by PCR and subcloned into the pAS2 vector (BD Biosciences). This bait plasmid was cotransformed together with a pretransformed human brain Matchmaker cDNA library in pACT-2 (BD Biosciences) into the CG1945 yeast strain. Clones growing in the absence of Leu, Trp, and His were picked, and their phenotypes were confirmed by β-galactosidase assay and retransformation. Plasmids from positive candidates were isolated and sequenced. Fusion Proteins and Antibodies—Expression of GST- and His6-tagged fusion proteins and purification using glutathione-Sepharose (AP Biotech, Freiburg, Germany) or nickel-nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) were performed as described by the manufacturers. Guinea pig anti-PIST antiserum was generated using a His6-fusion protein of PIST (amino acids 120–455; custom antiserum generation by Biogenes GmbH, Berlin, Germany). The rabbit anti-serum against the C terminus of the rat SSTR5 has been described before (19Stroh T. Kreienkamp H.J. Beaudet A. J. Comp. Neurol. 1999; 412: 69-82Crossref PubMed Scopus (69) Google Scholar). Anti-FLAG and anti-myc were from Sigma, and anti-T7 was from Novagen. Expression Constructs—The mouse SSTR5 cDNA was kindly provided by Wolfgang Meyerhof (Potsdam, Germany). Expression constructs for SSTR5 were generated by cloning PCR fragments coding for the cDNAs of human, rat, and mouse SSTR5 into a modified pcDNA3 vector (Invitrogen), which encodes an N-terminal T7-epitope tag (20Roth A. Kreienkamp H.J. Meyerhof W. Richter D. J. Biol. Chem. 1997; 272: 23769-23774Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The full-length coding regions of PIST and PEX5 (Peroxin 5), as well as residues 15–478 of human PDZK1 were cloned into pEGFP-C1. Full-length rat PDZK1 was obtained by reverse transcription-PCR from rat kidney and expressed as fusion with the myc-epitope tag in pcDNA3.1 (Invitrogen). Immunoprecipitation—Transfected were in of 5 on for were to of of were to the and for at all were in and on by were and on glutathione of protein for These were with of fusion protein in of for at were by and were of the with and were by using the growing on were with in and with in for at in for at were with anti-T7 or anti-myc in in or in in for at by of with or in was by using a as described (20Roth A. Kreienkamp H.J. Meyerhof W. Richter D. J. Biol. Chem. 1997; 272: 23769-23774Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, A. Kreienkamp H.J. D. Meyerhof W. Richter D. Biol. 1997; PubMed Scopus Google Scholar). were using AtT20 on were with at and to for in in were at with a of the in and pig anti-PIST in the were for at with to and pig to from in of of the and with to for the of on the interaction of PIST and SSTR5 in the AtT20 cell line, were for at in 5 with and before for various of with the to and as the were using and on a the of the were was using the and release By yeast two-hybrid screening in various cDNA from human or rat we identified interaction partners of the intracellular C-terminal of human and rodent SSTR5 PDZ domain protein interacting specifically with also as or G. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar, A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. J. M. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), a PDZ domain protein that is widely expressed in the brain and was found to be a potential interaction of the human as well as the rat SSTR5. the PDZ PIST of the second of which is by a A that the interaction PIST and SSTR5 is mediated by the PDZ domain We also identified the PDZ domain protein from kidney a protein with PDZ which is expressed in and 1998; Google Scholar). PDZK1 also with the C termini of rat and human The third protein we PEX5, also as the peroxisomal does not contain a PDZ domain a of these PEX5 has been to identify C-terminal target of proteins for sorting into The sequence for binding to PEX5 is G. C. A. P. D. 1995; PubMed Scopus Google Scholar), which is to the sequence for binding of C-terminal to type I PDZ proteins is a and Ref. M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1043) Google for The sequence in that the human not the rat SSTR5 to the sequence for also the mouse receptor C-terminal PEX5, as the C-terminal is in with in rat SSTR5. This was confirmed by a assay with a to the mouse SSTR5 C terminus not Thus the mouse and human receptors, which to the sequence for PEX5 PEX5, whereas the rat receptor does not to the and binding to PEX5. On the other rat and human receptors PIST and PDZK1 in a PDZ of interaction partners for the SSTR5 C-terminal tail used for yeast two-hybrid indicate sequence fragments used in the pAS2 bait The C-terminal residues from human, rat, and mouse are for of interaction partners of human and rat SSTR5. obtained from the yeast two-hybrid were by into yeast CG1945 using the bait vector or bait vector the C-terminal of rat or human SSTR5. In the case of PIST, constructs were also to the interacting as indicate the of the as by the of yeast to on not PDZ, PSD-95/discs interactions were in all cases confirmed in by using fusion of the C terminus of the rat and human as well as were and on the glutathione interacting partners were as His6-tagged and with the and by all PIST, PDZK1, and PEX5 well as an of were detected in the from the human not in the from the In PDZK1 and PIST also to the rat whereas the PEX5 the interaction of SSTR5 with this protein in the yeast two-hybrid In the case of PDZK1, we used this assay to the of PDZK1 that with the SSTR5. this constructs coding for of PDZK1 were used to the His6-tagged and used in the assay with the human fusion proteins that the third PDZ domain were by in this that interaction SSTR5 and PDZK1 is mediated by this domain We generated cell the mouse these or were with or as a were after of the receptor from cell using the were for the of the receptor and interacting proteins by In all receptor and interacting proteins were detected in and of PEX5, PIST, and PDZK1 was on the of the in In the case of PIST, we also the for the PDZ ligand motif at the C terminus of the receptor by the residues in the PIST was coexpressed with this receptor in it be with the that the interaction SSTR5 and PIST is mediated by a interaction the receptor C was to the PDZ domain-containing proteins PDZK1 and PIST because their interactions that of PEX5 and and were not to the human The subcellular distribution of expressed proteins was by the receptor was found at the cell surface when expressed PIST a as has been described before Ref. M. L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PDZK1 was found at the plasma membrane this is because of the interaction of PDZK1 with protein of Ref. S. D. P. A. L.A. A. J. H. Full Text Full Text PDF PubMed Scopus Google Scholar). PIST was into it was localized to a however, the distribution of the as most of the protein was found in an intracellular with the distribution of PIST In the which the PDZ ligand was not by PIST and was found at the cell surface of a PDZK1 plasmid into the cell line the distribution of the receptor that of the PDZ as colocalized at the plasma membrane Thus it that the of PDZ domain proteins the subcellular targeting of the determine these were to that we the of SSTR5 and PIST in mouse pituitary AtT20 which SSTR5 and have been frequently used to the of somatostatin on hormone of SSTR5 using a against the receptor C terminus that SSTR5 is localized in an intracellular identified as the by of its for P. Esdaile M.J. McPherson P.S. Schonbrunn A. Kreienkamp H.J. Beaudet A. J. Biol. Chem. 2004; 279: 8029-8037Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). with the PIST in pig that a of the SSTR5 with the for PIST, suggesting that proteins and interact in the Golgi/TGN in AtT20 We also these with for various of to signaling the distribution of the the of SSTR5 not these was the after of and Expression of the in these that the intracellular of the receptor is to the C-terminal PDZ ligand as the receptor was localized to the plasma with or intracellular to the of the interaction of the receptor with PDZ domain we the of PDZ domain on SSTR5 receptor has been widely that SSTR5 and many other are after agonist A. Kreienkamp H.J. D. Meyerhof W. Richter D. Biol. 1997; PubMed Scopus Google Scholar). the were with an agonist for the for the receptor was from the plasma membrane into an intracellular which be identified as the Golgi as it with the of the agonist an to and in to recycling of the receptor to the plasma membrane. this was performed on the which the PDZ binding the receptor was into a that the PDZ interaction is not required for the receptor the to be to the plasma membrane of PIST to a for the type as it was found largely in the Golgi as before in the absence of In the of PDZK1, on the other internalization of the receptor was this rather In that PDZK1 with the receptor into an intracellular We also the of PIST on receptor it that the PIST a recycling of the Thus PIST retains the receptor in the Golgi apparatus of its mammalian somatostatin receptor with the of a of SSTR2 M. M. G. V. FEBS Lett. PubMed Scopus Google Scholar), contain a potential C-terminal PDZ ligand the in the receptors for C from H.J. H.J. T. Hönck H.H. S. G. F. Richter D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), contain such a have that all interact with C-terminal binding and these interactions may the receptors to specific subcellular and signaling (9Zitzer H. Hönck H.H. Bächner D. Richter D. Kreienkamp H.J. J. Biol. Chem. 1999; 274: 32997-33001Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 10Schwärzler A. Kreienkamp H.J. Richter D. J. Biol. Chem. 2000; 275: 9557-9562Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The PDZ domain motif of SSTR5 from mouse and is as it also to the binding sequence for the peroxisomal receptor PEX5, and this protein has been in yeast two-hybrid In of SSTR5 on a peroxisomal targeting data Ref. G. S. B. A. F. J. Biol. PubMed Scopus Google Scholar) that the human and mouse receptors be to because of this we a interaction SSTR5 and PEX5 in we have not obtained for peroxisomal targeting of SSTR5 in these found the receptor at the plasma membrane not This that the C-terminal motif are required to target a membrane protein to On the other hand it may that PEX5 in signaling we have on the interactions of the which not be for human and mouse SSTR5 also for the rat Our data a interaction SSTR5 and the PDZ domains of PIST at the of the Golgi and of PDZK1 at the plasma The SSTR5 is in this most to of and the cystic fibrosis transmembrane of which also interact with PIST and PDZK1 their C-terminal PDZ ligand motifs M. L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). does also interact with the PDZ domain of regulatory factor R.A. M.J. Lefkowitz R.J. S. A. 1998; PubMed Scopus Google Scholar, S. M. FEBS Lett. 1998; PubMed Scopus Google Scholar), whereas the human SSTR5 does not A. Sondergaard B.P. Ersboll B. Holst B. Nielsen F.C. Haft C.R. Whistler J. Schwartz T.W. J. Biol. Chem. 2004; 279: 54291-54303Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). is found in the Golgi to PIST, whereas the at the membrane of Ref. M. L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the PDZ ligand motif is in the case of the to in the The C-terminal interaction may be of as that the of the PDZ ligand motif to intracellular and of the to that in cystic fibrosis M. C. J. D. J. S. M. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google Scholar, A. M. B. J. M. G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This was by other that a of the PDZ interaction in the regulation of B. C. D. J. J. S. A. PubMed Scopus Google Scholar, M. M. J. M. L. M. B. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). J. H. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) that PIST the for Here we that the PDZ ligand motif in the C terminus of the SSTR5 its intracellular the receptor is localized at the plasma membrane of AtT-20 its type which is found in the Golgi apparatus. Golgi is specific to the whereas the SSTR2 which is expressed in the which does not is found at the plasma membrane P. Esdaile M.J. McPherson P.S. Schonbrunn A. Kreienkamp H.J. Beaudet A. J. Biol. Chem. 2004; 279: 8029-8037Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The of the C-terminal motif in postendocytic sorting is to that of the binding motif in the as of this motif to a in receptor recycling A. M. B. J. M. G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). for the interaction of the C terminus with the regulatory factor PDZ domain may to recycling to the plasma membrane has been also for the Ref. D. A. Zastrow M. Nature. 1999; PubMed Scopus Google Scholar), we have for proteins PDZK1 and binding to the SSTR5 C terminus, suggesting that the of PIST and PDZK1 may determine the of the R.M. Zastrow M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have recently provided that type I PDZ are to recycling of the receptor of their to to exchanger regulatory we not PDZK1 in not we a model the receptor is to the Golgi after internalization of its PDZ ligand PIST the receptor in the Golgi/TGN and its sorting to the plasma membrane. its PIST associates with a protein that is in and the and in the sorting of membrane proteins such as the SSTR5 A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J.M. A. H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. L. S.R. J. R. Biol. 2004; PubMed Scopus Google Scholar). In this PIST be a sorting for a of transmembrane proteins from and the β1-adrenergic receptor; Ref. J. M. J. Hall R.A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) to cell molecules B. Schreff M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to channels or their proteins such as Y. J. Neurosci. 2004; 24: PubMed Scopus Google Scholar). PDZK1 also multiple of membrane This is in to proteins such as or the which appear to be specifically to the postendocytic sorting of recycling of the SSTR5 and targeting to the membrane from intracellular has been described T. P. C. Kreienkamp H.J. J. Beaudet A. Endocrinology. 2000; 141: PubMed Scopus Google Scholar). In it has been that of cell surface receptors targeting of receptors from the Golgi/TGN to the plasma membrane T. P. C. Kreienkamp H.J. J. Beaudet A. Endocrinology. 2000; 141: PubMed Scopus Google Scholar). is to that intracellular sorting by the interactions with PIST PDZK1 may to of receptors to the cell surface such be by the in its to of PIST to the plasma membrane J. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We to somatostatin be the to the SSTR5 to the cell surface in AtT-20 by activation of SSTR2 or the plasma membrane SSTR5. we to the cell be to as the of also to we that of receptor to the cell surface have a on the of somatostatin to regulate growth hormone secretion from the pituitary or insulin secretion from the We Hönck and for and for PIST

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