Title: Trans-inactivation of Receptor Tyrosine Kinases by Novel Angiotensin II AT2 Receptor-interacting Protein, ATIP
Abstract: Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic β2 receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade. Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic β2 receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade. The potent vasoactive peptide angiotensin II (Ang II) 1The abbreviations used are: Ang II, angiotensin II; bFGF, basic fibroblast growth factor; CHO, Chinese hamster ovary; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; PDGF, platelet-derived growth factor; RTK, receptor tyrosine kinase; VSMC, vascular smooth muscle cell; ATIP-ID, AT2 receptor-interacting protein-(interacting domain); HA, hemagglutinin. is also an important regulator of cellular proliferation and hypertrophy. This peptide binds to two main subtypes of receptors (AT1 and AT2) that both belong to the superfamily of G protein-coupled receptors (GPCR) but display opposite biological and physiological effects. The AT1 receptor mediates most of the known cardiovascular and central actions of Ang II. This subtype has mitogenic and trophic effects in many tissues and cell types and transduces multiple intracellular signaling cascades typically associated with GPCR activation. In contrast, AT2 behaves like a “natural antagonist” of the AT1 subtype on most physiological functions, and induces anti-proliferative and proapoptotic effects in vitro and in vivo (for reviews, see Refs. 1Horiuchi M. Lehtonen J. Daviet L. Trends Endocrinol. Metab. 1999; 10: 391-396Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 2Nouet S. Nahmias C. Trends Endocrinol. Metab. 2000; 11: 1-6Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 3Gallinat S. Busche S. Raizada M. Sumners C. Am. J. Physiol. 2000; 278: 357-374Crossref PubMed Google Scholar, 4Stoll M. Unger T. Regul. Pept. 2001; 99: 175-182Crossref PubMed Scopus (103) Google Scholar, 5Volpe M. Musumeci B. Paolis P.D. Savoia C. Morganti A. J. Hypertens. 2003; 21: 1429-1443Crossref PubMed Scopus (96) Google Scholar). The AT2 receptor activates unconventional signaling pathways that in most cases do not involve coupling to classical regulatory G proteins. A growing body of evidence indicates that anti-growth effects of the AT2 receptor are associated with activation of tyrosine phosphatases and inhibition of protein kinases, which ultimately lead to inhibition of extracellular regulated kinase (ERK2). In many cell types, AT2 is functionally coupled to the Src homology 2 domain-containing tyrosine phosphatase SHP-1 (6Bedecs K. Elbaz N. Sutren M. Masson M. Susini C. Strosberg A.D. Nahmias C. Biochem. J. 1997; 325: 449-454Crossref PubMed Scopus (202) Google Scholar, 7Lehtonen J. Daviet L. Nahmias C. Horiuchi M. Dzau V. Mol. Endocrinol. 1999; 13: 1051-1060Crossref PubMed Scopus (69) Google Scholar, 8Cui T. Nakagami H. Iwai M. Takeda Y. Shiuchi T. Daviet L. Nahmias C. Horiuchi M. Cardiovasc. Res. 2001; 49: 863-871Crossref PubMed Scopus (70) Google Scholar). This phosphatase has been shown to play a central role in the AT2 signaling cascades leading to inhibition of AT1-induced PYK2 and Jun kinase (9Matsubara H. Shibasaki Y. Okigaki M. Mori Y. Masaki H. Kosaki A. Tsutsumi Y. Uchiyama Y. Fujiyama S. Nose A. Iba O. Tateishi E. Hasegawa T. Horiuchi M. Nahmias C. Iwasaka T. Biochem. Biophys. Res. Commun. 2001; 282: 1085-1089Crossref PubMed Scopus (36) Google Scholar), AT1-trans-activated EGF receptor tyrosine kinase (10Shibasaki Y. Matsubara H. Nozawa Y. Mori Y. Masaki H. Kosaki A. Tsutsumi Y. Uchiyama Y. Fujiyama S. Nose A. Iba O. Tateishi E. Hasegawa T. Horiuchi M. Nahmias C. Iwasaka T. Hypertension. 2001; 38: 367-372Crossref PubMed Scopus (45) Google Scholar), and insulin-induced phosphatidylinositol 3-kinase and Akt activation (11Cui T. Nakagami H. Nahmias C. Shiuchi T. Takeda-Matsubara Y. Li J. Wu L. Iwai M. Horiuchi M. Mol. Endocrinol. 2002; 16: 2113-2123Crossref PubMed Scopus (51) Google Scholar). AT2 negatively cross-talks with receptor tyrosine kinases (RTK) such as bFGF, EGF, and insulin receptors (10Shibasaki Y. Matsubara H. Nozawa Y. Mori Y. Masaki H. Kosaki A. Tsutsumi Y. Uchiyama Y. Fujiyama S. Nose A. Iba O. Tateishi E. Hasegawa T. Horiuchi M. Nahmias C. Iwasaka T. Hypertension. 2001; 38: 367-372Crossref PubMed Scopus (45) Google Scholar, 12Elbaz N. Bedecs K. Masson M. Sutren M. Strosberg A.D. Nahmias C. Mol. Endocrinol. 2000; 14: 795-804Crossref PubMed Scopus (56) Google Scholar, 13DePaolis P. Porcellini A. Savoia C. Lombardi A. Gigante B. Frati G. Rubattu S. Musumeci B. Volpe M. J. Hypertens. 2002; 20: 693-699Crossref PubMed Scopus (30) Google Scholar) by targeting a very early step of RTK activation, i.e. autophosphorylation of the receptor. In vascular smooth muscle cells (VSMC) from AT2-transgenic mice, EGF receptor trans-inactivation induced by AT2 stimulation was found to involve rapid activation of tyrosine phosphatase SHP-1 and its increased association with the EGF receptor (10Shibasaki Y. Matsubara H. Nozawa Y. Mori Y. Masaki H. Kosaki A. Tsutsumi Y. Uchiyama Y. Fujiyama S. Nose A. Iba O. Tateishi E. Hasegawa T. Horiuchi M. Nahmias C. Iwasaka T. Hypertension. 2001; 38: 367-372Crossref PubMed Scopus (45) Google Scholar). In Chinese hamster ovary (CHO) cells, however, AT2-mediated trans-inactivation of the insulin receptor does not involve protein dephosphorylation by orthovanadate-sensitive tyrosine phosphatases nor coupling to pertussis toxin-sensitive regulatory heterotrimeric Gi/Go proteins (12Elbaz N. Bedecs K. Masson M. Sutren M. Strosberg A.D. Nahmias C. Mol. Endocrinol. 2000; 14: 795-804Crossref PubMed Scopus (56) Google Scholar), suggesting that another yet undefined mechanism may link AT2 receptor stimulation to growth inhibition. A number of recent studies have revealed that GPCRs can mediate their intracellular effects through signaling pathways that are independent of G proteins (14Marinissen M. Gutkind J. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar, 15Brady A. Limbird L. Cell. Signal. 2002; 14: 297-309Crossref PubMed Scopus (224) Google Scholar). Over the past few years, many groups have identified novel intracellular proteins that directly interact with C-terminal tails of GPCRs and function as scaffolds to regulate receptor trafficking or signaling (16Hall R. Lefkowitz R. Circ. Res. 2002; 91: 672-680Crossref PubMed Scopus (186) Google Scholar, 17Bockaert J. Marin P. Dumuis A. Fagni L. FEBS Lett. 2003; 546: 65-72Crossref PubMed Scopus (190) Google Scholar). ATRAP is one such example of a novel protein that selectively interacts with the AT1 receptor C terminus and down-regulates its activity (18Daviet L. Lehtonen J. Tamura K. Griese D. Horiuchi M. Dzau V. J. Biol. Chem. 1999; 274: 17058-17062Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 19Cui T. Nakagami H. Iwai M. Takeda Y. Shiuchi T. Tamura K. Daviet L. Horiuchi M. Biochem. Biophys. Res. Commun. 2000; 279: 938-941Crossref PubMed Scopus (71) Google Scholar). Regarding the AT2 receptor, recent studies have documented a direct interaction of its C-terminal tail with ErbB3, a member of the EGF receptor family (20Knowle D. Ahmed S. Pulakat L. Regul. Pept. 2000; 87: 73-82Crossref PubMed Scopus (28) Google Scholar, 21Pulakat L. Gray A. Johnson J. Knowle D. Burns V. Gavini N. FEBS Lett. 2002; 524: 73-78Crossref PubMed Scopus (18) Google Scholar), and with the transcription factor promyelocytic zinc finger containing protein (PLZF) abundantly expressed in the heart (22Senbonmatsu T. Saito T. Landon E.J. Watanabe O. Price Jr., E. Roberts R.L. Imboden H. Fitzgerald T.G. Gaffney F.A. Inagami T. EMBO J. 2003; 22: 6471-6482Crossref PubMed Scopus (173) Google Scholar). In the present study, we have used the C-terminal part of the AT2 receptor as bait in a two-hybrid system to identify new interacting partners of the receptor. We describe here the molecular cloning and functional characterization of ATIP1, a novel coiled-coil domain containing protein that selectively interacts with the AT2 receptor and mediates inhibition of growth factor-induced ERK2 activation and cell proliferation. Yeast Two-hybrid and cDNA Cloning—The 52 C-terminal residues of the human AT2 receptor were PCR amplified and subcloned into pGBT9 vector in-frame with the Gal4-DNA binding domain. Independent transformants (3 × 106) from a mouse fetal cDNA library containing inserts of 300 to 700 bp in VP16 vector (a kind gift of Dr. A. Vojtek) were screened by the two-hybrid system cloning method in the HF7 strain as described (23Vojtek A. Sm S.H. Cooper J. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1717) Google Scholar) using the 52 C-terminal residues of the AT2 receptor as a bait. A 354-bp cDNA clone designated ATIP was repeatedly isolated from two independent screenings of the library. Specificity of the interaction was verified with pGBT9 vectors containing lamin or RAS (kind gifts of Dr. J. Camonis) as unrelated baits. cDNA fragments corresponding to the 65 C-terminal residues of the human bradykinin B2 receptor (kind gift of Dr. W. Müller-Esterl), the last 86 residues of the human β2-adrenergic receptor (kindly provided by Dr. R. Jockers), or the last 62 residues of the rat AT1 receptor (a kind gift of Dr. K. Bernstein) were PCR amplified and subcloned into pGBT9 to assay for selectivity of the interaction. For isolation of full-length murine mATIP1 cDNA, the 354-bp ATIP insert was used to screen a mouse fetal cDNA library (3.5 × 105 colonies) of large inserts constructed into the pcDNA1 expression vector (24Nahmias C. Cazaubon S. Briend-Sutren M. Lazard D. Villageois P. Strosberg A.D. Biochem. J. 1995; 306: 87-92Crossref PubMed Scopus (56) Google Scholar). For isolation of the human hATIP1 homolog, the 3′ region (last 755 bp) of mATIP1 cDNA was used to screen 3 × 105 colonies of a human lung cDNA library in pcDNA3 (Invitrogen). For isolation of full-length human hATIP2 cDNA, reverse transcriptase-PCR amplification was performed on poly(A+) from human myometrium with specific oligonucleotides: 5′-cgggatccgtatccagggctcatgttcacttg-3′ (sense) and 5′-ccgctcgagtgctgatatacctcttgtgcccac-3′ (antisense). The resulting cDNA fragment (1.37 kb) was subcloned into the BamHI and XhoI sites of the pcDNA3 vector (Invitrogen) and entirely sequenced. Cell Lines and Transfections—Chinese hamster ovary cells expressing the human AT2 receptor (CHO-hAT2 cells) have been described elsewhere (12Elbaz N. Bedecs K. Masson M. Sutren M. Strosberg A.D. Nahmias C. Mol. Endocrinol. 2000; 14: 795-804Crossref PubMed Scopus (56) Google Scholar). These cells were stably transfected using Dosper liposomal transfection reagent (Roche Diagnostics) with the 354-bp ATIP-ID cDNA fragment subcloned into pcDNA3 (clones C11 and C12), or full-length mATIP1 cDNA in pcDNA3 (clones L11 and L14), or pcDNA3 vector alone (clones V11 and V13). Positive clones resistant to G418 (800 μg/ml) were analyzed by immunoblotting using anti-ATIP polyclonal antibodies. AT2 receptor binding sites were analyzed by radioligand binding performed on whole cells (8 × 104 cells/well in 24-well dishes) using 0.25 nm [125I-Sar,Ile]Ang II (PerkinElmer Life Sciences) in the presence or absence of Ang II (1 μm) or CGP 42112 (1 μm), as described (25Akishita M. Ito M. Lehtonen J. Daviet L. Dzau V. Horiuchi M. J. Clin. Investig. 1999; 103: 63-71Crossref PubMed Scopus (88) Google Scholar). All clones were used at passages 5 to 20. COS-hAT2 cells permanently expressing the human AT2 receptor have been previously described (26Lazard D. Briend-Sutren M. Villageois P. Mattei M. Strosberg A.D. Nahmias C. Recept. Channels. 1994; 2: 271-280PubMed Google Scholar) and were used at passages 3 to 16. Adult rat VSMC were previously shown to express both AT1 and AT2 receptors (25Akishita M. Ito M. Lehtonen J. Daviet L. Dzau V. Horiuchi M. J. Clin. Investig. 1999; 103: 63-71Crossref PubMed Scopus (88) Google Scholar). These cells were prepared as described (25Akishita M. Ito M. Lehtonen J. Daviet L. Dzau V. Horiuchi M. J. Clin. Investig. 1999; 103: 63-71Crossref PubMed Scopus (88) Google Scholar) and used at passages 2 to 8. Immunoprecipitations and Immunoblotting—The entire coding regions of mATIP1, hATIP1, and hATIP2 were fused in-frame with the Myc epitope, or the HA epitope inserted N-terminal into the pcDNA3 expression vector. For dimerization studies, COS-1 cells (1.6 × 106 cells per 100-mm dish) were transfected with 5 μg of appropriate tagged cDNA using 6 μl of FuGENE (Roche) as described by the manufacturer. Forty-eight hours after transfection, cell lysates (1% Triton X-100, 50 mm Hepes, 150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, pH 7.5) were immunoprecipitated using anti-Myc monoclonal antibody 9E10 (10 μg) covalently coupled to agarose (Santa Cruz Biotechnology). Associated proteins were eluted in urea-SDS sample buffer (62 mm Tris, pH 6.8, 2% SDS, 2,5% β-mercaptoethanol, 6 m urea, 20% glycerol, bromphenol blue solution) for 15 min at 60 °C and resolved on 10% SDS-PAGE gels. Western blotting was carried out as described (6Bedecs K. Elbaz N. Sutren M. Masson M. Susini C. Strosberg A.D. Nahmias C. Biochem. J. 1997; 325: 449-454Crossref PubMed Scopus (202) Google Scholar) using anti-HA polyclonal antibodies (Santa Cruz). Membranes were stripped and reblotted with rabbit anti-ATIP polyclonal antibodies. For co-immunoprecipitation experiments, CHO-hAT2 cells stably transfected with either pcDNA3 or full-length mATIP1-Myc were starved in serum-free medium for 24 h and treated for 5 min at 37 °C with or without Ang II (100 nm). Cell lysates (500 μg proteins) were immunoprecipitated using 1 μg of polyclonal rabbit anti-AT2 antibody (sc-9040, Santa Cruz) overnight. Western blots were revealed with anti-Myc monoclonal antibody 9E10 (Santa Cruz). Membranes were stripped and reprobed with goat anti-AT2 antibodies (sc-7421, Santa Cruz) for internal control. For analyzing expression of endogenous ATIP proteins, whole lysates were prepared as described (27Di Liberto G. Dallot E. Eude-Le-Parco I. Cabrol D. Ferre F. Breuiller-Fouche M. Am. J. Physiol. 2003; 285: C599-C607Crossref PubMed Scopus (31) Google Scholar) from human tissues (kindly provided by Dr. Pascal Pineau, Institut Pasteur, Paris, and Dr. Michelle Breuiller-Fouche, Institut Cochin, Paris) and human cell lines. Proteins (20 μg) were separated on a 10% SDS-PAGE and immunoblotted using rabbit anti-ATIP polyclonal antibodies (1:5000). Production of Rabbit Anti-ATIP Polyclonal Antibodies—The 354-bp ATIP-ID cDNA fragment was subcloned into the pRSETA vector (Invitrogen) and the resulting polypeptide: His6-ATIP fused to six histidine residues, was purified from bacterial lysates by passage through a nickel column as described by the manufacturer. Purified His6-ATIP (100 μg) was injected three times intradermally into rabbits at 2-week intervals for production of polyclonal antiserum. Anti-ATIP polyclonal antibodies were affinity purified by passage through glutathione-agarose beads coupled to the glutathione S-transferase-ATIP fusion protein (obtained by subcloning the 354-bp ATIP-ID insert into pGEX-4T1 (Amersham Biosciences) and purification of glutathione S-transferase-ATIP as described by the manufacturer). Specificity of anti-ATIP antibodies was verified by immunoblotting cell lysates from COS cells transfected with each ATIP cDNA. A single polypeptide migrating at the expected molecular weight (18,000, 55,000, 55,000, and 50,000 for ATIP-ID, mATIP1, hATIP1, and hATIP2, respectively) was detected in each case. Measurement of ERK2 Phosphorylation—Stably transfected CHO-hAT2 cells (clones L11, L14, V11, V13, C11, and C12) were seeded at a density of 2 × 105 cells/well in 6-well dishes and treated as described (12Elbaz N. Bedecs K. Masson M. Sutren M. Strosberg A.D. Nahmias C. Mol. Endocrinol. 2000; 14: 795-804Crossref PubMed Scopus (56) Google Scholar). Total cell lysates were analyzed by immunoblotting with polyclonal anti-phospho-ERK antibodies (Cell Signaling). Blots were reprobed with monoclonal anti-ERK2 antibodies (UBI) as an internal control, and further incubated with monoclonal anti-phosphotyrosine antibodies (4G10, UBI). Alternatively, total cell lysates were submitted to 10% SDS-PAGE and immunoblotted under conditions (6Bedecs K. Elbaz N. Sutren M. Masson M. Susini C. Strosberg A.D. Nahmias C. Biochem. J. 1997; 325: 449-454Crossref PubMed Scopus (202) Google Scholar) that allow to visualize the activated, slower migrating form of endogenous ERK2. Transient Expression and Phosphorylation of Tagged ERK2—For measurement of ERK2 phosphorylation in transient transfections, COS-wt or COS-hAT2 cells were seeded at a density of 3 × 105 cells/well in 6-well dishes and transfected with mATIP1 cDNA or empty vector (1 μg) in 3 μl of FuGENE (Roche) as indicated by the manufacturer. To avoid high background because of stimulation of endogenous ERK2 in non-transfected cells, co-transfections were performed with 0.5 μg of a construct (“ERK2-Myc”) encoding ERK2 fused to six Myc epitopes (a kind gift of Dr. Sabine Traver, Paris). The resulting ERK2-Myc polypeptide migrates at higher molecular weight (60,000) and can thus be easily distinguished from endogenous ERK2 (42,0000) in Western blot. Forty-eight hours after transfection, cells were starved in serum-free Dulbecco's modified Eagle's medium for 18 h before appropriate treatment with EGF as indicated (12Elbaz N. Bedecs K. Masson M. Sutren M. Strosberg A.D. Nahmias C. Mol. Endocrinol. 2000; 14: 795-804Crossref PubMed Scopus (56) Google Scholar), then lysed in 60 μl of Laemmli's sample buffer and analyzed by immunoblotting (20 μl) with anti-phospho-ERK antibodies (Cell Signaling). Membranes were stripped and reblotted with anti-Myc antibodies (Santa Cruz) to assess expression levels of ERK2-Myc in each lane. Measurement of Thymidine Incorporation—DNA synthesis was assayed by measuring [3H]thymidine incorporation as described (25Akishita M. Ito M. Lehtonen J. Daviet L. Dzau V. Horiuchi M. J. Clin. Investig. 1999; 103: 63-71Crossref PubMed Scopus (88) Google Scholar). Stably transfected CHO-hAT2 cells (clones V11, V13, L11, and L14) were seeded at a density of 2 × 105 cells/well in 24-well dishes (60% density) in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. On the following day, cells were set in serum-free medium for 24 h to induce quiescence. For VSMC, cells were seeded at a density of 7 × 104 cells/well in 24-well dishes and transfected with mATIP1 cDNA or empty vector (0.4 μg/well). Forty-eight hours after transfection, cells (80–90% confluency) were cultured for 48 h in serum-free medium to induce quiescence. For measurement of DNA synthesis, quiescent cells were treated with the indicated growth factor for 40 h and pulsed with 1 μCi/ml [3H]thymidine (PerkinElmer Life Sciences) for an additional 24 h. Cells were washed with ice-cold phosphate-buffered saline and treated as described previously (25Akishita M. Ito M. Lehtonen J. Daviet L. Dzau V. Horiuchi M. J. Clin. Investig. 1999; 103: 63-71Crossref PubMed Scopus (88) Google Scholar) to measure radioactivity of the cell lysate. Molecular Cloning of ATIP1 from Mouse and Man—The yeast two-hybrid system cloning method was developed to identify novel interacting partners of the AT2 receptor. The last 52 amino acids of the human AT2 receptor were used as a bait to screen 3 × 106 clones of a mouse fetal cDNA library constructed in the pVP16 cloning vector (23Vojtek A. Sm S.H. Cooper J. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1717) Google Scholar). One positive clone containing an insert of 354 bp was repeatedly isolated from two independent screenings of the library. This insert encoded an open reading frame of 118 amino acids that was designated ATIP-ID, for AT2 receptor-interacting protein-(interacting domain). As shown in Fig. 1A, ATIP-ID interacts with the C-terminal intracellular tail of the AT2 receptor but not with that of the AT1 receptor subtype, nor with C-terminal domains of other GPCR such as β2-adrenergic or bradykinin B2 receptors. The interaction between ATIP-ID and the AT2 C terminus was further confirmed by in vitro binding assays using purified domains of each protein (data not shown). The 354-bp fragment of ATIP-ID was used as a probe to screen a mouse fetal cDNA library constructed in the pcDNA1 expression vector. A full-length cDNA clone of 1803 nucleotides was isolated (GenBank™ accession number AF173380). This clone contains an open reading frame of 1323 bp translating into a 440-amino acid polypeptide that we have designated mouse ATIP1 (mATIP1) (Fig. 1B). Several in-frame stop codons were found upstream of the initiating methionine. The human homolog of mATIP1 was further isolated by screening a human adult lung cDNA library, using as a probe a 755-bp cDNA fragment corresponding to the 3′ end of the mATIP1 coding sequence. Full-length human ATIP1 (hATIP1) cDNA encompasses 1977 nucleotides (GenBank™ accession number AF293357) and encodes a 436-amino acid polypeptide that shares 86% amino acid sequence identity with mATIP1. While this manuscript was in revision, a nucleotide sequence (MTSG1) identical to hATIP1 was published in the data banks (GenBank™ accession number AF121259). Mouse and human ATIP1 proteins are mainly hydrophilic and contain no transmembrane domain. The major part of ATIP1 is composed of a large coiled-coil domain (residues 106 to 375 of mATIP1) including two leucine zippers (Fig. 1B). ATIP1 is also characterized by a high proportion of basic residues (16%) and a stretch of 30 C-terminal residues rich in proline, serine/threonine, and arginine (“PSR” region). A BLAST search for homologous proteins in the data banks indicated that ATIP1 is a novel protein that shares 25% amino acid sequence identity with myosins in the coiled-coil region. Immunoprecipitation experiments were carried out to investigate whether ATIP1 was also able to interact with the AT2 receptor in a cellular context. Full-length mATIP1 cDNA was fused to the Myc epitope and transfected into CHO cells expressing the AT2 receptor. Co-immunoprecipitation experiments (Fig. 1C) revealed constitutive interaction between ATIP1 and AT2, which is not significantly modified upon treatment with Ang II. A Family of Homologous ATIP Proteins—We then analyzed the tissue distribution of ATIP1 mRNA. The 354-bp fragment of ATIP-ID was used as a probe to hybridize a Northern blot of mRNA from various human tissues (Fig. 2A). Expression of a 1.9-kb transcript likely corresponding to hATIP1 mRNA was detected in all tissues examined. Additional hybridizing transcripts were detected at 4.2 and 6.9 kb in spleen, prostate, ovary, small intestine, colon (Fig. 2A), as well as in heart, placenta, skeletal muscle, pancreas, and lung (data not shown), suggesting the existence of additional mRNAs homologous to ATIP1. Western blot analysis of total lysates from human tissues and cell lines using a polyclonal antibody directed against the ATIP-ID domain (Fig. 2A, right panel) further confirmed endogenous expression of hATIP1-like proteins. Four major polypeptides of apparent molecular weights 30,000, 60,000, 120,000, and 180,000, respectively, were expressed at variable levels in different tissues. Further work will be required to unambiguously identify each of these polypeptides, and determine the relationship between the various mRNA transcripts and polypeptides detected with the ATIP-ID probe and specific antibodies, respectively. Nucleotide sequence comparisons with GenBank™ data bases allowed the identification of human cDNA sequences related to hATIP1 in uterus (accession number AL096842), brain (partial sequence, accession number AB033114), and fetal brain (accession number AK125188). All three sequences contained the 354-bp sequence of ATIP-ID and were therefore designated hATIP2 (uterus), hATIP3 (brain), and hATIP4 (fetal brain). Close examination of genomic sequences in the data bases revealed that all four hATIP mRNAs are derived from a single gene by alternative promoter utilization and exon/intron splicing. 2M. Di Benedetto, manuscript in preparation. Translated amino acid sequences of hATIP2, hATIP3, and hATIP4 comprise 415, 1270, and 517 residues, respectively, and are 100% identical to hATIP1 in their last 395 amino acids (Fig. 2B). It is of note that the C-terminal sequence shared by all four hATIP members includes the large coiled-coil domain and leucine zippers as well as the stretch of 118 amino acids (ATIP-ID) that interact with AT2. In the N terminus, hATIP proteins differ both in length and sequence, and exhibit specific motifs that suggest differential intracellular localizations and/or association with distinct cytosolic partners (Fig. 2B). hATIP2 thus exhibits a very short (20 amino acids) N-terminal region that contains a bipartite nuclear localization signal, and one polyproline-rich region (PPXXP) known to play an important role in protein-protein interactions with WW or Src homology 3 domains (28Macias M. Wiesner S. Sudol M. FEBS Lett. 2002; 513: 30-37Crossref PubMed Scopus (398) Google Scholar). hATIP3 has a long N-terminal region (874 residues) containing one nuclear localization signal and four polyproline-rich motifs (Fig. 2B). The N-terminal part of hATIP4 (122 amino acids) contains a stretch of 24 hydrophobic amino acids flanked on each side by a charged residue, which is a typical feature of membrane spanning regions. hATIP4 thus likely consists of a transmembrane protein with a short (36 residues) N-terminal extracellular domain and two polyproline-rich motifs at the inner face and close vicinity of the membrane (Fig. 2B). Dimerization of ATIP Proteins—The presence of a large coiled-coil domain with two leucine zippers in the C-terminal part of all ATIP proteins suggested that these proteins may be able to dimerize. Co-immunoprecipitation experiments were undertaken to investigate this possibility. The cDNAs encoding hATIP1 and hATIP2 were fused either to the Myc or HA epitopes, and co-transfected into COS cells prior to immunoprecipitation with anti-Myc antibodies. As seen in Fig. 3 (left panel), HA-hATIP2 was revealed in anti-Myc immunoprecipitates from cells co-transfected with Myc-hATIP2 and HA-hATIP2 (lane 3) but not from cells transfected either with Myc-hATIP2 or HA-hATIP2 alone (first and fourth lanes), therefore indicating that hATIP2 homodimerizes inside the cell. Similarly, HA-hATIP1 was detected in anti-Myc immunoprecipitates from COS cells co-transfected with Myc-hATIP1 and HA-hATIP1 (Fig. 3, right panel, fourth lane), but not from cells transfected with either Myc-hATIP1 or HA-hATIP1 alone (first and third lanes), therefore indicating homodimerization of hATIP1. Finally, the ability of hATIP1 and hATIP2 to heterodimerize was demonstrated by co-immunoprecipitation of HA-hATIP1 and Myc-hATIP2 using anti-Myc antibodies (Fig. 3, right panel, second lane). Inhibitory Effect of mATIP1 on Growth Factor-induced ERK2 Activation—We then sought to examine the functional role of ATIP1, and we first analyzed whether ectopic expression of ATIP1 in eukaryotic cells was able either to block, or mimic, AT2 receptor activation. Our previous studies had demonstrated negative cross-talk between AT2 and growth factor receptors, leading to an inhibitory effect of AT2 receptor on insulin-ind