Somatostatin Receptor Interacting Protein Defines a Novel Family of Multidomain Proteins Present in Human and Rodent Brain

By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile α motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA. By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile α motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA. PSD-95/discs large/ZO-1 cortactin-binding protein 1 glutathioneS-transferase human embryonic kidney postsynaptic density sterile α motif synapse-associated protein 102 Src homology 3 somatostatin receptor somatostatin receptor interacting protein kilobase pairs Targeting of neurotransmitter receptors to postsynaptic or presynaptic sites is an area that has been widely studied in recent years; a large body of evidence has accumulated showing that many receptors are anchored at their specifc site of action by specialized anchoring proteins, which may link receptors to components of the synaptic structure or the cytoskeleton (1Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1626) Google Scholar, 2Kuhse J. Betz H. Kirsch J. Curr. Opin. Neurobiol. 1995; 5: 318-323Crossref PubMed Scopus (188) Google Scholar). This is true for inhibitory as well as excitatory receptors of the family of ligand-gated ion channels. For the second large family of neurotransmitter receptors, the seven transmembrane domain G-protein-coupled receptors, only very recently have some proteins been identified that may be involved in anchoring or linkage to the cytoskeleton. These include the homer proteins, which are tightly associated with metabotropic glutamate receptors via a PDZ1 domain in homer and the C terminus of the mGluRs (3Brakeman P.R. Lanahan A.A. O'Brien R. Roche K. Barnes C.A. Huganir R.L. Worley P.F. Nature. 1997; 386: 284-288Crossref PubMed Scopus (929) Google Scholar). However, for the large majority of G-protein-coupled receptors, no intracellular associated proteins have been identified so far beyond those proteins which are necessary for signal transduction and functional regulation of the receptors,i.e. the G-proteins and proteins of the arrestin family (4Lefkowitz R.J. Inglese J. Koch W.J. Pitcher J. Attramadal H. Caron M. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 127-133Crossref PubMed Google Scholar). We have begun to address this issue for members of the somatostatin receptor family (SSTRs). SSTRs are widely expressed in neuronal tissue and modulate synaptic responses by interacting with inhibitory G-proteins in presynaptic as well as postsynaptic compartments of neurons (e.g. Refs. 5Boehm S. Betz H. J. Neurosci. 1997; 17: 4066-4075Crossref PubMed Google Scholar, 6Dournaud P. Gu Y.Z. Schonbrunn A. Mazella J. Tannenbaum G.S. Beaudet A. J. Neurosci. 1996; 16: 4468-4478Crossref PubMed Google Scholar, 7Dournaud P. Boudin H. Schonbrunn A. Tannenbaum G.S. Beaudet A. J. Neurosci. 1998; 18: 1056-1071Crossref PubMed Google Scholar, 8Schulz S. Schreff M. Schmidt H. Handel M. Przewlocki R. Höllt V. Eur. J. Neurosci. 1998; 10: 3700-3708Crossref PubMed Scopus (98) Google Scholar). Recently we have used the yeast two-hybrid system to screen for proteins intracellularly associated with SSTR2, one of the major SSTR subtypes in the mammalian brain. Here we show that the C terminus of SSTR2, which contains the consensus sequence for recognition of PDZ domains, interacts with a novel protein termed SSTR interacting protein or SSTRIP. The latter defines a novel family of multidomain cytoskeletal anchoring proteins that are highly enriched in the postsynaptic density fraction derived from rat brain. A yeast two-hybrid screen using the rat SSTR2 C terminus as a bait in the Gal4-DNA binding domain vector pAS2 was described previously (9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The initial partial clone of 900 base pairs (clone 16; Ref. 9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) coding for the PDZ domain of SSTRIP was used as a probe to screen human fetal brain, adult thalamus, and hippocampus cDNA libraries (CLONTECH) in order to obtain the full-length sequence of SSTRIP. By using oligonucleotide primers based on the human sequence, a partial sequence was also amplified from rat hypothalamic cDNA and used for screening of a rat brain cDNA library (kindly provided by Dr. Rainer Reinscheid). A cDNA fragment encoding the PDZ domain of human SSTRIP was cloned into the GST fusion protein vector pGEX2T (Amersham Pharmacia Biotech) and used for the generation of fusion protein. Overlay assays with a biotinylated fusion protein containing the C terminus of SSTR2 were performed as described (9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The construction of an SSTR2-cDNA carrying an N-terminal T7 epitope tag in the expression vector pcDNA3 (Invitrogen, Leek, The Netherlands) has been described before (10Roth 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, 11Schwartkop C.-P. Kreienkamp H.-J. Richter D. J. Neurochem. 1999; 72: 1275-1282Crossref PubMed Scopus (31) Google Scholar). Ntag-SSTR2 and SSTRIP were coexpressed in human embryonic kidney (HEK) cells by transient transfection using the calcium phosphate method as described (12Kreienkamp H.-J. Maeda R. Sine S.M. Taylor P. J. Biol. Chem. 1994; 269: 8108-8114Abstract Full Text PDF PubMed Google Scholar). For immunoprecipitations, cells were lyzed in 1 ml of RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mmNaCl, 50 mm Tris-HCl, pH 7.4) on ice for 20 min and centrifuged at 15,000 × g for 20 min. The epitope-tagged receptor was precipitated from the supernatant fraction using the monoclonal T7-antibody (3 μg) and protein A-Sepharose (Amersham Pharmacia Biotech; 100 μl of a 50% suspension in RIPA buffer) as described (11Schwartkop C.-P. Kreienkamp H.-J. Richter D. J. Neurochem. 1999; 72: 1275-1282Crossref PubMed Scopus (31) Google Scholar). Precipitates were washed five times with RIPA buffer and denatured by boiling in Laemmli sample buffer. After separation by polyacrylamide gel electrophoresis, proteins were blotted onto nitrocellulose membranes. SSTRIP was then detected by immunoblotting using the anti-PDZ domain antibody as a primary antibody and goat anti-rabbit coupled to alkaline phosphatase as secondary antibody. Whole brains of adult rats were frozen on dry ice, and 20-μm sections were prepared on a cryostat (Leitz, Wetzlar, Germany). Antisense RNA probes labeled with α-35S-UTP (NEN Life Science Products) were generated using the rat SSTR2 cDNA (13Kluxen F. Bruns C. Lübbert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4618-4622Crossref PubMed Scopus (194) Google Scholar) and a rat SSTRIP-clone matching bases 1544–2619 of the human SSTRIP sequence. In situhybridization experiments were performed as described (14Bettler B. Boulter J. Hermans-Borgmeyer I. O'Shea-Greenfield A. Deneris E.S. Moll C. Borgmeyer U. Hollmann M. Heinemann S. Neuron. 1990; 5: 583-595Abstract Full Text PDF PubMed Scopus (512) Google Scholar). Sections were exposed to BioMax MR film for 3 days. For higher resolution studies, the same slides were dipped into Kodak NTB-2 nuclear emulsion, developed in Dektol developer (Eastman Kodak Co.) after 2–3 weeks of exposure, and subsequently stained with Giemsa (Sigma). Rat brain membranes were fractionated according to the method described by Carlin et al. (15Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (605) Google Scholar). Briefly, a postnuclear membrane fraction (crude membranes) was applied to a sucrose multistep gradient, and the synaptosomal fraction was recovered at the interface between 1.0 and 1.2 m sucrose after centrifugation at 82,500 ×g for 2 h. After extraction with 1% Triton X-100, the PSD-one Triton fraction was obtained in a second ultracentrifugation step at 200,000 × g at the interface between 1.5 and 2.0 m sucrose and used for Western blotting experiments. We used the C terminus of the rat SSTR2 as a bait in a yeast two-hybrid screen for potentially interacting proteins (9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); one novel clone of 900 base pairs, termed SSTRIP for somatostatin receptor interacting protein, was obtained from a human cDNA library which interacted specifically with SSTR2 when compared with the empty bait vector or nonrelated control proteins. This clone encodes a protein fragment that contains a PDZ domain which is highly similar to that of cortactin-binding protein 1 (CortBP1; see Refs. 9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar and 16Du Y. Weed S.A. Xiong W.C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (219) Google Scholar). Screening of rat and human brain cDNA libraries yielded several splice variants of the full-length SSTRIP as depicted in Fig.1. The longest cDNA derived from human brain contains an open reading frame of 6546 base pairs, coding for a protein of 240 kDa. In this protein, the PDZ domain is preceded by the SH3 domain and a domain containing six ankyrin repeats. The PDZ domain is followed by seven proline-rich domains that may function as SH3-binding domains (17Sudol M. Oncogene. 1998; 17: 1469-1474Crossref PubMed Scopus (195) Google Scholar). At the C terminus a sterile α motif (SAM domain) is observed which has been implicated as a dimerization motif for transcription factors as well as receptor tyrosine kinases (18Thanos C.D. Goodwill K.E. Bowie J.U. Science. 1999; 283: 833-836Crossref PubMed Scopus (200) Google Scholar,19Stapleton D. Balan I. Pawson T. Sicheri F. Nat. Struct. Biol. 1999; 6: 44-49Crossref PubMed Scopus (213) Google Scholar). A splice variant of SSTRIP was observed in which the N-terminal region with the six ankyrin repeats as well as the SH3 domain is replaced by the alternative N terminus labeled as exon a in Fig. 1. Accordingly, a major splice site appears to be located between the SH3 domain and the PDZ domain, as an additional partial clone was observed which contains the SH3 and the PDZ domains but lacks an insert of 9 amino acids (insert b in Fig. 1) at this position. Alternative splicing C-terminal of the PDZ domain leads to transcripts that contain an additional insert of 8 amino acids (exon cin Fig. 1) between the PDZ and the first proline-rich domain. However, this variant was so far detected only in rat tissues. In order to confirm the interaction observed in the yeast system in terms of true protein-protein interactions, we performed an overlay assay with a GST fusion protein of the PDZ domain of SSTRIP. Fusion proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes; a biotinylated fusion protein of the SSTR2 C terminus was used as a probe, according to Ref. 9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar. In these experiments, the PDZ domain of SSTRIP exhibited a strong interaction with SSTR2, whereas the GST control protein did not react (Fig.2 A). In vivoattempts to coimmunoprecipitate SSTR2 and SSTRIP from a plasma membrane fraction of rat brains were not successful due to the fact that SSTRIP could only be solubilized in the presence of RIPA supplemented with 2% SDS. These conditions did not permit the detection of specific interactions between SSTR2 and SSTRIP although immunoreactivity of the individual proteins was still present in the solubilized membrane fraction (data not shown). To circumvent this technical problemin vitro, coexpression experiments were performed in HEK cells. An expression vector containing the N-terminal part of SSTRIP (amino acids 1–1288), including the ankyrin, SH3, PDZ, and some of the proline-rich regions, was cotransfected with the rat SSTR2 carrying a N-terminal T7 epitope tag (NT7-SSTR2). When SSTR2 was precipitated from cellular lysates using the T7 antibody, SSTRIP could be efficiently detected in the precipitate by Western blotting. SSTRIP could not be detected in the precipitate when SSTR2 was omitted from the transfection mixture, indicating that SSTR2 specifically interacts with SSTRIP in these cells (Fig. 2 B). It should be noted that SSTRIP coexpressed with the inward rectifier potassium channel protein Kir 2.1 known to carry the consensus sequence for the PDZ domain and to bind to PSD95 (20Cohen N.A. Brenman J.E. Snyder S.H. Bredt D.S. Neuron. 1996; 17: 759-767Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) in HEK cells did not show coimmunoprecipitation from cell lysates (data not shown) excluding the possibility that SSTRIP interacts with any protein carrying the PDZ consensus sequence at its C terminus. The specificity of the interaction between the SSTR2 C terminus and the PDZ domain of SSTRIP was further analyzed in the yeast two-hybrid system. Bait plasmids encoding the C termini of SSTR1–4 as well as a C-terminally mutated SSTR2 (the last amino acid residue isoleucine is replaced by seven amino acids derived from the bait vector sequence) were coexpressed with the initial target vector obtained from the human brain cDNA library. Activation of the His-3 that on was observed only in the presence of the SSTR2 to a in the presence of the C terminus was when the SSTR2 C terminus was These were by a assay on indicating that the interaction between SSTR2 and the PDZ domain an C terminus A interaction of with the domain that was detected could not be in an overlay (data not specifically interacts with the SSTR2 C SSTR on W. H.-J. C. Richter D. Cell Biol. 10: PubMed Scopus Google (13Kluxen F. Bruns C. Lübbert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4618-4622Crossref PubMed Scopus (194) Google additional bases from the expression F. Bruns C. Lübbert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4618-4622Crossref PubMed Scopus (194) Google W. I. C. S. Richter D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Y. J. M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google C termini of rat SSTR1–4 as well as a mutated SSTR2 in which the last amino an is replaced by seven amino acid residues were cloned into the bait vector were cotransfected with the human derived from the initial yeast two-hybrid screen into the S. and on no the of were observed with the SSTR2 very were obtained with the not The are in additional bases from the expression in a The C termini of rat SSTR1–4 as well as a mutated SSTR2 in which the last amino an is replaced by seven amino acid residues were cloned into the bait vector were cotransfected with the human derived from the initial yeast two-hybrid screen into the S. and on no the of were observed with the SSTR2 very were obtained with the not The are in by of RNA from human the SSTRIP is expressed in the human brain, in the and transcripts of 9 and are detected the latter to one of the splice variants of SSTRIP in Fig. 1. in brain as or is to the this may that splice variants of SSTRIP are expressed in brain. In order to the expression of SSTR2 and we performed in experiments of rat brain in Fig. in a striking overlap in the expression of SSTR2 and SSTRIP was including of the and the proteins are also expressed in the A major between SSTR2 and SSTRIP was in the are in the and the SSTRIP mRNA was detected not only in cell bodies but also in the molecular layer, suggesting that SSTRIP to the of which are into neuronal (Fig. A fusion protein containing the PDZ domain was used to due to the sequence between and the SSTRIP protein in this domain, these proteins from rat and human with similar This was used in Western blotting experiments on rat brain membrane specific were observed at molecular from to (Fig. from rat brain at an molecular of Y. Weed S.A. Xiong W.C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (219) Google suggesting that the at higher molecular to the splice variants of SSTRIP. for as well as for SSTRIP was obtained in a membrane fraction as well as in the synaptosomal fraction and in postsynaptic PSD-one Triton are enriched in the PSD-one Triton fraction when compared with the membrane fraction and the synaptosomal fraction. This is similar to that observed in a with an antibody specific for synapse-associated protein 102 which has been characterized as a major of postsynaptic U. S. W.J. S. Huganir R.L. Neuron. 1996; 17: Full Text Full Text PDF PubMed Scopus Google Scholar). To the that SSTRIP is enriched in the density we used a second antibody the ankyrin domain of SSTRIP. In this only a at 240 was observed which is also enriched in the fraction (Fig. show that the domain of SSTRIP is in one of the several variants of the of which are enriched in the postsynaptic density fraction. Here we the of human a protein that interacts specifically with the C terminus of the SSTR2 via a PDZ the of from rat brain termed and have been in the base which strong homology to the human SSTRIP protein and are in fact to partial clone from Rat and a domain structure to human whereas the rat sequence lacks the domain. In a protein termed associated protein 1) was C. W. H. K. J. J. Neurosci. 1999; PubMed Google Scholar) which is to but which several alternative splicing sites in to those for SSTRIP. The homology between SSTRIP and is to the PDZ and domains, the sequence between the proteins and In the region between the PDZ and the domain a sequence of A cDNA may be as SSTRIP and to a fragment from the human with very sequence in the PDZ and the PDZ domains of these proteins are very appears that the many variants of these proteins may be for anchoring SSTR2 in many in This is in with which have that the SSTR2 protein is present in presynaptic as well as in postsynaptic compartments of neuronal cells P. Gu Y.Z. Schonbrunn A. Mazella J. Tannenbaum G.S. Beaudet A. J. Neurosci. 1996; 16: 4468-4478Crossref PubMed Google Scholar, 8Schulz S. Schreff M. Schmidt H. Handel M. Przewlocki R. Höllt V. Eur. J. Neurosci. 1998; 10: 3700-3708Crossref PubMed Scopus (98) Google Scholar). The domain structure and splicing when SSTRIP and (9Zitzer H. Richter D. Kreienkamp H.-J. J. Biol. Chem. 1999; 274: 18153-18156Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Du Y. Weed S.A. Xiong W.C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (219) Google Scholar, C. W. H. K. J. J. Neurosci. 1999; PubMed Google Scholar) suggests that these proteins a family of multidomain proteins. It to be the ankyrin domain and the SH3 domain are also present in splice variants of and the protein derived from human The strong of proteins of this family in the postsynaptic density fraction from rat brain that we have detected an of this postsynaptic In this the function of SSTRIP and its may far beyond an anchoring function for somatostatin receptors, as family members have been identified by for interaction for Y. Weed S.A. Xiong W.C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (219) Google or the protein for the rat of SSTRIP in the see Refs. M. Y. K. A. M. Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar and S. A. A. M. J. Cell Biol. 1997; PubMed Scopus Google Scholar). interaction have been for the PDZ domains of the family of proteins J. Curr. Opin. Neurobiol. 1999; PubMed Scopus Google which bind glutamate receptor as well as potassium channel and many proteins. yeast two-hybrid experiments to additional proteins that with the PDZ domains as well as with the ankyrin, SH3, and domains of SSTRIP. We and Hermans-Borgmeyer for with the in experiments and Dr. for the antibody. in the of this the rat of SSTRIP has been as by and J. Biol. Chem. and J. R. P. and M. and as by and Y. J. Biol. Chem.