Immunophilins in the Nervous System
Authors: Solomon H Snyder, Michael M Lai, Patrick E Burnett
Journal: Neuron (1998)

The term immunophilin is used to designate receptors for immunosuppressant drugs such as cyclosporin A (CsA), FK506, and rapamycin. In clinical medicine, immunosuppression is used therapeutically for a variety of purposes. One of the most important is the treatment of patients undergoing organ transplantation to prevent host-versus-graft rejection (71Morris P.J Cyclosporine, FK-506 and other drugs in organ transplantation.Curr. Opin. Immunol. 1991; 3: 748-751Crossref PubMed Scopus (29) Google Scholar, 41Fung J.J Starzl T.E FK 506 in solid organ transplantation.Transplant. Proc. 1994; 26: 3017-3020PubMed Google Scholar). Immunosuppression also diminishes symptoms of autoimmune disease such as myocarditis and glomerulonephritis (100Thomson A.W Starzl T.E FK 506 and autoimmune disease perspective and prospects.Autoimmunity. 1992; 12: 303-313Crossref PubMed Scopus (24) Google Scholar). Historically, immunosuppression was achieved with drugs that inhibit cell division, as lymphocytes tend to divide more rapidly than mature somatic cells. Accordingly, mitotic inhibitors employed in cancer chemotherapy, such as methotrexate, have been extensively employed as immunosuppressants, but with significant toxicity. In contrast, modern immunosuppressants, of which the first widely employed agent was CsA, demonstrate a reasonable degree of selectivity for the immune system. Modern immunosuppressant drugs are also valuable tools for elucidating intracellular signaling systems. Many of these drugs act at very low nanomolar concentrations and so display great selectivity. CsA and FK506 have clarified how calcineurin, the calcium-dependent phosphatase, regulates the entry of transcription factors into the nucleus. Rapamycin has elucidated novel signaling systems that regulate protein translation. CsA, FK506, and rapamycin are large molecules first isolated from microbial fermentation broths (Figure 1). CsA and FK506 cause immunosuppression by blocking the calcium-dependent activation of antigen-reactive T cells (10Borel J.F Feurer C Gubler H.U Steahelin H Biological effects of cyclosporine A a new antilymphocytic agent.Agents Actions Suppl. 1976; 6: 468-475Crossref Scopus (1171) Google Scholar, 59Kay J.E Benzie C.R Borghetti A.F Effect of cyclosporine A on lymphocyte activation by the calcium ionophore A23187.Immunology. 1983; 50: 441-446PubMed Google Scholar). Rapamycin inhibits the clonal proliferation of T cells by preventing signaling through the interleukin-2 (IL-2) receptor (7Bierer B.E Mattila P.S Standaert R.F Herzenberg L.A Burakoff S.J Crabtree G Schreiber S.L Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.Proc. Natl. Acad. Sci. USA. 1990; 87 (a): 9231-9235Crossref PubMed Scopus (606) Google Scholar, 9Bierer B.E Jin Y.J Fruman D.A Calvo V Burakoff S.J FK 506 and rapamycin molecular probes of T-lymphocyte activation.Transplant. Proc. 1991; 23: 2850-2855PubMed Google Scholar). Rapamycin is also an antimitotic agent toward a large number of cell types, and because of this, it has been explored as an anticancer agent (24Chen H Wu J Xu D Luo H Daloze P Efficacy and mechanism of action of rapamycin in presensitized recipients of experimental allografts.Transplant. Proc. 1992; 24: 1669-1670PubMed Google Scholar). The greatest advances in understanding how these drugs act came with the identification of the immunophilins as their receptors. Initial research was restricted to investigations in lymphocytes. Appreciation that concentrations of the immunophilins in the brain are 10–50 times greater than levels in the immune system (93Steiner J.P Dawson T.M Fotuhi M Glatt C.E Snowman A.M Cohen N Snyder S.H High brain densities of the immunophilin FKBP colocalized with calcineurin.Nature. 1992; 358: 584-587Crossref PubMed Scopus (316) Google Scholar) has led to a rapidly developing body of evidence indicating important neural roles for the immunophilins. The first important insight into how immunosuppressants act came with the observation by Handschumacher and associates (1984) that CsA binds to a cytoplasmic 18 kDa protein designated cyclophilin A. Subsequently, numerous members of the cyclophilin family, which generally have larger molecular weights, have been identified (92Stamnes M.A Zuker C.S Peptidyl-prolyl cis-trans isomerases, cyclophilin, FK506-binding protein, and ninaA four of a kind.Curr. Opin. Cell Biol. 1990; 2: 1104-1107Crossref PubMed Scopus (15) Google Scholar). The second member, designated cyclophilin B, is 22 kDa and is localized together with calreticulin to calciosomes, calcium-storing organelles of the endoplasmic reticulum (76Price E.R Zydowsky L.D Jin M.J Baker C.H McKeon F.D Walsh C.T Human cyclophilin B a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence.Proc. Natl. Acad. Sci. USA. 1991; 88: 1903-1907Crossref PubMed Scopus (271) Google Scholar). The 23 kDa cyclophilin C is greatly enriched in the kidney, where it is localized to the endoplasmic reticulum (40Friedman J Weissman I Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin one in the presence and one in the absence of CsA.Cell. 1991; 66: 799-806Abstract Full Text PDF PubMed Scopus (356) Google Scholar). Cyclophilin 40 is a component of the inactive form of the glucocorticoid receptor and is believed to function as a chaperone in this context (39Freeman B.C Toft D.O Morimoto R.I Molecular chaperone machines chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.Science. 1996; 274: 1718-1720Crossref PubMed Scopus (284) Google Scholar). A large body of research had established that the actions of FK506 and CsA on the immune system function were essentially identical, with both drugs inhibiting the early, calcium-dependent phases involved in IL-2 formation. Hence, it was hypothesized that they share a common receptor. Remarkably, FK506 has no affinity for any of the cyclophilins. Binding studies with [3H]FK506 revealed that it interacts instead with a small soluble protein designated FK506 binding protein (FKBP) (49Harding M.W Galat A Uehling D.E Schreiber S.L A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.Nature. 1989; 341: 758-760Crossref PubMed Scopus (1155) Google Scholar, 87Siekierka J.J Staruch M.J Hung S.H Sigal N.H FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein, which is distinct from the cyclosporin A-binding protein, cyclophilin.J. Immunol. 1989; 143 (b): 1580-1583PubMed Google Scholar). The FKBPs demonstrate no amino acid sequence homology nor any major conformational resemblance to the cyclophilins. Like the cyclophilins, the FKBPs comprise a large family, whose most abundant and first identified member is FKBP12, a 12 kDa soluble protein (49Harding M.W Galat A Uehling D.E Schreiber S.L A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.Nature. 1989; 341: 758-760Crossref PubMed Scopus (1155) Google Scholar, 87Siekierka J.J Staruch M.J Hung S.H Sigal N.H FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein, which is distinct from the cyclosporin A-binding protein, cyclophilin.J. Immunol. 1989; 143 (b): 1580-1583PubMed Google Scholar, 58Kay J.E Structure-function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases.Biochem. J. 1996; 314: 361-385Crossref PubMed Scopus (146) Google Scholar). FKBP13 displays about 40% amino acid sequence identity to FKBP12 but is localized to the lumen of the endoplasmic reticulum (57Jin Y.J Albers M.W Lane W.S Bierer B.E Schreiber S.L Burakoff S.J Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13.Proc. Natl. Acad. Sci. USA. 1991; 88: 6677-6681Crossref PubMed Scopus (155) Google Scholar). FKBP25 is a nuclear protein that interacts with casein kinase II, as well as nucleolin, a nucleolar protein that participates in ribosome formation (42Galat A Lane W.S Standaert R.F Schreiber S.L A rapamycin-selective 25-kDa immunophilin.Biochemistry. 1992; 31: 2427-2434Crossref PubMed Scopus (114) Google Scholar, 56Jin Y.J Burakoff S.J The 25-kDa FK506-binding protein is localized in the nucleus and associates with casein kinase II and nucleolin.Proc. Natl. Acad. Sci. USA. 1993; 90: 7769-7773Crossref PubMed Scopus (111) Google Scholar). FKBP52 (also designated FKBP59 or hsp56), like cyclophilin-40, is part of the glucocorticoid receptor complex, to which it is linked via the 90 kDa heat shock protein (97Tai P.K Albers M.W Chang H Faber L.E Schreiber S.L Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex.Science. 1992; 256: 1315-1318Crossref PubMed Scopus (263) Google Scholar). Additional family members that have not been as well characterized are FKBP12.6 (64Lam E Martin M.M Timerman A.P Sabers C Fleischer S Lukas T Abraham R.T O'Keefe S.J O'Neill E.A Wiederrecht G.J A novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor.J. Biol. Chem. 1995; 270: 26511-26522Crossref PubMed Scopus (163) Google Scholar) and FKBP51-FKBP54 (88Smith D.F Albers M.W Schreiber S.L Leach K.L Deibel Jr., M.R FKBP54, a novel FK506-binding protein in avian progesterone receptor complexes and HeLa extracts.J. Biol. Chem. 1993; 268: 24270-24273Abstract Full Text PDF PubMed Google Scholar, 4Baughman G Wiederrecht G.J Campbell N.F Martin M.M Bourgeois S FKBP51, a novel T-cell–specific immunophilin capable of calcineurin inhibition.Mol. Cell. Biol. 1995; 15: 4395-4402Crossref PubMed Scopus (143) Google Scholar). Since rapamycin influences different components of the immune response than FK506 and CsA, most investigators assumed that it would have a unique receptor. Surprisingly, rapamycin binds to FKBP12 with greater affinity than FK506 (33Dumont F.J Melino M.R Staruch M.J Koprak S.L Fischer P.A Sigal N.H The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells.J. Immunol. 1990; 144 (a): 1418-1424PubMed Google Scholar, 34Dumont F.J Staruch M.J Koprak S.L Melino M.R Sigal N.H Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin.J. Immunol. 1990; 144 (b): 251-258PubMed Google Scholar, 9Bierer B.E Jin Y.J Fruman D.A Calvo V Burakoff S.J FK 506 and rapamycin molecular probes of T-lymphocyte activation.Transplant. Proc. 1991; 23: 2850-2855PubMed Google Scholar). The peculiar pattern of drug interactions with the two immunophilin families initially stymied efforts to explain immunosuppressive effects. Researchers looked for some common property. The first insight was that cyclophilin A has enzymatic activity, catalyzing the isomerization of peptide proline residues between cis and trans conformations, an action that should influence protein folding (38Fischer G Wittmann-Liebold B Lang K Kiefhaber T Schmid F.X Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins.Nature. 1989; 337: 476-478Crossref PubMed Scopus (1172) Google Scholar, 98Takahashi N Hayano T Suzuki M Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin.Nature. 1989; 337: 473-475Crossref PubMed Scopus (913) Google Scholar). CsA inhibits this peptidyl prolyl isomerase, or rotamase, activity at nanomolar concentrations. FKBP12 also possesses rotamase activity, which is inhibited by FK506 and rapamycin (49Harding M.W Galat A Uehling D.E Schreiber S.L A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.Nature. 1989; 341: 758-760Crossref PubMed Scopus (1155) Google Scholar, 86Siekierka J.J Hung S.H Poe M Lin C.S Sigal N.H A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin.Nature. 1989; 341 (a): 755-757Crossref PubMed Scopus (857) Google Scholar). Thus, rotamase inhibition might appear fundamental to immunosuppression. However, numerous compounds that bind to the immunophilins and inhibit their rotamase activity lack immunosuppressant effects (8Bierer B.E Somers P.K Wandless T.J Burakoff S.J Schreiber S.L Probing immunosuppressant action with a nonnatural immunophilin ligand.Science. 1990; 250 (b): 556-559Crossref PubMed Scopus (291) Google Scholar, 35Dumont F.J Staruch M.J Koprak S.L Siekierka J.J Lin C.S Harrison R Sewell T Kindt V.M Beattie T.R Wyvratt M et al.The immunosuppressive and toxic effects of FK-506 are mechanistically related pharmacology of a novel antagonist of FK-506 and rapamycin.J. Exp. Med. 1992; 176: 751-760Crossref PubMed Scopus (228) Google Scholar). Moreover, these immunosuppressive drugs typically act at low nanomolar concentrations, whereas tissue levels of some immunophilins are almost micromolar, so that in intact tissues, only a very small portion of rotamase activity would be inhibited (83Schreiber S.L Chemistry and biology of the immunophilins and their immunosuppressive ligands.Science. 1991; 251: 283-287Crossref PubMed Scopus (1304) Google Scholar). In principle, these immunosuppressants might act via one of the immunophilins whose tissue density is very low. Alternatively, one might hypothesize a "gain-of-function" mechanism whereby the drug–immunophilin complex would bind to a second protein that could not be influenced by the immunophilin or drug alone. The first drug–immunophilin target was discovered by 66Liu J Farmer Jr., J.D Lane W.S Friedman J Weissman I Schreiber S.L Calcineurin is a common target of cyclophilin–cyclosporin A and FKBP–FK506 complexes.Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3468) Google Scholar and 40Friedman J Weissman I Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin one in the presence and one in the absence of CsA.Cell. 1991; 66: 799-806Abstract Full Text PDF PubMed Scopus (356) Google Scholar, who showed that complexes of cyclophilin A–CsA and FKBP12–FK506 bind to calcineurin, a calcium/calmodulin-activated protein phosphatase, and inhibit its activity. The relative potencies of FK506, CsA, and related compounds in inhibiting calcineurin correlate closely with their immunosuppressive potencies, suggesting a role for calcineurin in their immunosuppressive action. In T cells, an important calcineurin substrate is the phosphorylated form of the transcription factor family, nuclear factor of activated T cells (NF-AT). The NF-ATs stimulate the transcription of a number of genes required for T cell proliferation, notably IL-2 (101Tocci M.J Matkovich D.A Collier K.A Kwok P Dumont F Lin S Degudicibus S Siekierka J.J Chin J Hutchinson N.I The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes.J. Immunol. 1989; 143: 718-726PubMed Google Scholar). However, the NF-AT protein cannot enter the nucleus unless it is dephosphorylated by calcineurin. Therefore, the mechanism of immunosuppression by these drugs is to form a drug–immunophilin complex that inhibits the calcineurin phosphatase activity and prevents nuclear translocation of the NF-ATs (Figure 2). How can rapamycin bind to FKBP12 yet differ from FK506 in its immunologic effects? The observation that rapamycin could compete with FK506 for its intracellular receptor led to the discovery that rapamycin and FK506 shared FKBP12 as a common receptor (7Bierer B.E Mattila P.S Standaert R.F Herzenberg L.A Burakoff S.J Crabtree G Schreiber S.L Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.Proc. Natl. Acad. Sci. USA. 1990; 87 (a): 9231-9235Crossref PubMed Scopus (606) Google Scholar, 33Dumont F.J Melino M.R Staruch M.J Koprak S.L Fischer P.A Sigal N.H The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells.J. Immunol. 1990; 144 (a): 1418-1424PubMed Google Scholar, 34Dumont F.J Staruch M.J Koprak S.L Melino M.R Sigal N.H Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin.J. Immunol. 1990; 144 (b): 251-258PubMed Google Scholar). Unlike FK506, the FKBP12–rapamycin complex does not bind or inhibit calcineurin. Because low nanomolar concentrations of rapamycin block signaling in cells that contain micromolar concentrations of FKBP12, most investigators assume that rapamycin, like FK506, acts by a gain-of-function mechanism, with the FKBP12–rapamycin complex binding to another target protein. To seek such a target, we and others looked for proteins that might bind to FKBP12 only in the presence of rapamycin. We identified two proteins that interact selectively with radiolabeled FKBP12 only when rapamycin is present and cloned the cDNA for the larger one, designated rapamycin and FKBP12 target 1 (RAFT1) (79Sabatini D.M Erdjument-Bromage H Lui M Tempst P Sndyer S.H RAFT1 a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1145) Google Scholar). Independently, Schreiber and colleagues (13Brown E.J Albers M.W Shin T.B Ichikawa K Keith C.T Lane W.S Schreiber S.L A mammalian protein targeted by G1-arresting rapamycin–receptor complex.Nature. 1994; 369: 756-758Crossref PubMed Scopus (1570) Google Scholar) cloned the cDNA for the same protein and called it FKBP and rapamycin associated protein (FRAP). RAFT1/FRAP has subsequently been identified by other workers (26Chiu M.I Katz H Berlin V RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex.Proc. Natl. Acad. Sci. USA. 1994; 91: 12574-12578Crossref PubMed Scopus (388) Google Scholar, 82Sabers C.J Martin M.M Brunn G.J Williams J.M Dumont F.J Wiederrecht G Abraham R.T Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells.J. Biol. Chem. 1995; 270: 815-822Crossref PubMed Scopus (657) Google Scholar). RAFT1 is the mammalian homolog of two yeast proteins, Tor1 and Tor2 (target of rapamycin) (51Heitman J Movva N.R Hall M.N Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.Science. 1991; 253: 905-909Crossref PubMed Scopus (1418) Google Scholar, 61Kunz J Henriquez R Schneider U Deuter-Reinhard M Movva N.R Hall M.N Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression.Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (693) Google Scholar), which when mutated confer resistance to the lethal effects of rapamycin in yeast. Accordingly, RAFT1 is also referred to as mammalian Tor (mTor). RAFT1 contains a C-terminal area that is homologous to the kinase domain of phosphatidylinositol-3 kinase (PI-3 kinase) (13Brown E.J Albers M.W Shin T.B Ichikawa K Keith C.T Lane W.S Schreiber S.L A mammalian protein targeted by G1-arresting rapamycin–receptor complex.Nature. 1994; 369: 756-758Crossref PubMed Scopus (1570) Google Scholar, 79Sabatini D.M Erdjument-Bromage H Lui M Tempst P Sndyer S.H RAFT1 a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1145) Google Scholar) and is the founding mammalian member of a new family of proteins that includes the DNA-activated protein kinase (DNA-PKcs) (50Hartley K.O Gell D Smith G.C Zhang H Divecha N Connelly M.A Admon A Lees-Miller S.P Anderson C.W Jackson S.P DNA-dependent protein kinase catalytic subunit a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product.Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (657) Google Scholar) and the protein product of the gene that is disrupted in the disease ataxia telangiectasia (ATM) (65Lavin M.F Khanna K.K Beamish H Spring K Watters D Shiloh Y Relationship of the ataxia-telangiectasia protein ATM to phosphoinositide 3 kinase.Trends Biochem. Sci. 1995; 20: 382-383Abstract Full Text PDF PubMed Scopus (77) Google Scholar). While RAFT1 and the yeast TOR proteins have lipid kinase activity (21Cardenas M.E Heitman J FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity.EMBO J. 1995; 14: 5892-5907Crossref PubMed Scopus (108) Google Scholar, 80Sabatini D.M Pierchala B.A Barrow R.K Schell M.J Snyder S.H The rapamycin and FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity.J. Biol. Chem. 1995; 270: 20875-20878Crossref PubMed Scopus (64) Google Scholar), it is unclear whether this enzymatic activity is intrinsic to RAFT1/Tor or a tightly associated lipid kinase. Rapamycin's interference with protein translation is thought to be the mechanism for its antimitotic activity. The drug blocks several steps in growth factor actions on translational regulators. Recent work in our laboratory and others' has demonstrated that RAFT1 functions as a protein kinase that phosphorylates several key regulators of protein translation (Figure 3). One of the shared effects of mitogenic stimuli is a rapid phosphorylation of the ribosome, primarily on the S6 protein of the 40S subunit. Phosphorylation of the S6 protein increases the ribosome's affinity for a subset of mRNAs containing significant 5′ secondary structure or polypyrimidine tracts (55Jefferies H.B Reinhard C Kozma S.C Thomas G Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family.Proc. Natl. Acad. Sci. USA. 1994; 91: 4441-4445Crossref PubMed Scopus (542) Google Scholar, 99Terada N Patel H.R Takase K Kohno K Nairn A.C Gelfand E.W Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins.Proc. Natl. Acad. Sci. USA. 1994; 91: 11477-11481Crossref PubMed Scopus (314) Google Scholar). Rapamycin blocks S6 phosphorylation by p70 S6 kinase (28Chung J Kuo C.J Crabtree G.R Blenis J Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (988) Google Scholar, 62Kuo C.J Chung J Fiorentino D.F Flanagan W.M Blenis J Crabtree G.R Rapamycin selectively inhibits interleukin--2 activation of p70 S6 kinase.Nature. 1992; 358: 70-73Crossref PubMed Scopus (548) Google Scholar). Moreover, we found that the p70 S6 kinase is directly phosphorylated by RAFT1, and this phosphorylation can be inhibited by rapamycin (15Burnett P.E Barrow R.K Cohen N.A Snyder S.H Sabatini D.M RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.Proc. Natl. Acad. Sci. USA. 1998; 95 (a): 1432-1437Crossref PubMed Scopus (896) Google Scholar). Therefore, one mechanism of translational inhibition by rapamycin is directly inhibiting RAFT1-dependent ribosomal phosphorylation. All mRNAs have a 7-methylguanosine cap structure at their 5′-end that mediates translation initiation. This cap is bound by eukaryotic initiation factor (eIF-4E). To initiate translation, eIF-4E must also bind the eIF-4G protein and thereby recruit the eIF-4F complex to the mRNA (91Sonenberg, N. (1996). mRNA 5′ CAP-binding protein EIF-4E and control of cell growth. In Translational Control, J.W.B. Hershey, M.B. Matthews, and N. Sonenberg, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 245–269.Google Scholar). This process can be inhibited by the unphosphorylated form of the 4E binding protein (4E-BP1), also known as Phas-1. When 4E-BP1 is phosphorylated by RAFT1 (14Brunn G.J Hudson C.C Sekulic A Williams J.M Hosoi H Houghton P.J Lawrence J.C.J Abraham R.T Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.Science. 1997; 277: 99-101Crossref PubMed Scopus (771) Google Scholar, 15Burnett P.E Barrow R.K Cohen N.A Snyder S.H Sabatini D.M RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.Proc. Natl. Acad. Sci. USA. 1998; 95 (a): 1432-1437Crossref PubMed Scopus (896) Google Scholar) and other mitogen-stimulated kinases, it can no longer bind to eIF-4E, and the translation initiation complex is assembled (5Beretta L Gingras A.C Svitkin Y.V Hall M.N Sonenberg N Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation.EMBO J. 1996; 15: 658-664Crossref PubMed Scopus (582) Google Scholar, 103von Manteuffel S.R Gingras A.C Ming X.F Sonenberg N Thomas G 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.Proc. Natl. Acad. Sci. USA. 1996; 93: 4076-4080Crossref PubMed Scopus (219) Google Scholar). Rapamycin inhibits the phosphorylation of 4E-BP1 by RAFT1 and therefore inhibits the initiation of protein translation (14Brunn G.J Hudson C.C Sekulic A Williams J.M Hosoi H Houghton P.J Lawrence J.C.J Abraham R.T Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.Science. 1997; 277: 99-101Crossref PubMed Scopus (771) Google Scholar, 15Burnett P.E Barrow R.K Cohen N.A Snyder S.H Sabatini D.M RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.Proc. Natl. Acad. Sci. USA. 1998; 95 (a): 1432-1437Crossref PubMed Scopus (896) Google Scholar). In cellular homogenates, RAFT1 is partially soluble and partially particulate, and so its activity might be regulated by translocation between the cytoplasm and the membrane. In a yeast two-hybrid analysis, we discovered that RAFT1 interacts with gephyrin (81Sabatini, D.M., Blackshaw, S., Barrow, R.K., Fields, M., Kirsh, J., Betz, H., and Snyder, S.H. (1996). RAFT-1 interactions with the clustering protein gephyrin are required for rapamycin-sensitive signaling. In NEN Symposium on Signal Transduction (Boston: NEN DuPont), pp. 5–6.Google Scholar), first identified as a protein that copurifies with the receptor for the neurotransmitter glycine (6Betz H Kuhse J Fischer M Schmieden V Laube B Kuryatov A Langosch D Meyer G Bormann J Rundstrom N et al.Structure, diversity and synaptic localization of inhibitory glycine receptors.J. Physiol. (Paris). 1994; 88: 243-248Crossref PubMed Scopus (40) Google Scholar). Gephyrin interacts with a domain of RAFT1 that displays considerable homology to the gephyrin binding site of the glycine receptor. The binding of RAFT1 to gephyrin is crucial for its phosphorylation of p70 S6 kinase and 4E-BP1 because mutations in RAFT1 that block its binding to gephyrin also block its ability to regulate p70 S6 kinase and 4E-BP1 (81Sabatini, D.M., Blackshaw, S., Barrow, R.K., Fields, M., Kirsh, J., Betz, H., and Snyder, S.H. (1996). RAFT-1 interactions with the clustering protein gephyrin are required for rapamycin-sensitive signaling. In NEN Symposium on Signal Transduction (Boston: NEN DuPont), pp. 5–6.Google Scholar). The discovery that immunosuppressants bind with high affinity to immunophilins and stimulate interactions with calcineurin implies the existence of endogenous ligands for the immunophilins that somehow influence calcineurin activity. However, no evidence has yet been obtained for an endogenous small molecule ligand for immunophilins. Moreover, the fact that immunosuppressant drugs influence calcineurin activity does not necessarily mean that immunophilins normally interact with calcineurin. Evidence for a physiological association of immunophilins and calcineurin has come from studies of brain localizations. Initial studies monitoring [3H]FK506 binding showed that FKBP12 levels in immune tissues such as the thymus resemble levels in other peripheral tissues such as the spleen, heart, kidney, liver, and lung. In contrast, binding levels in the brain are up to 50 times higher than in any peripheral tissue, depending on the brain region examined (93Steiner J.P Dawson T.M Fotuhi M Glatt C.E Snowman A.M Cohen N Snyder S.H High brain densities of the immunophilin FKBP colocalized with calcineurin.Nature. 1992; 358: 584-587Crossref PubMed Scopus (316) Google Scholar, 31Dawson T.M Steiner J.P Lyons W.E Fotuhi M Blue M Snyder S.H The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain relationship to calcineurin.Neuroscience. 1994; 62: 569-580Crossref PubMed Scopus (124) Google Scholar). The more than 10-fold variation in [3H]FK506 binding levels among various brain regions has been corroborated by localization of its mRNA by in situ hybridization (93Steiner J.P Dawson T.M Fotuhi M Glatt C.E Snowman A.M Cohen N Snyder S.H High brain densities of the immunophilin FKBP colocalized with calcineurin.Nature. 1992; 358: 584-587Crossref PubMed Scopus (316) Google Scholar). FKBP12 is sufficiently enriched in neurons that extraneuronal levels are not detected routinely. Highest densities of the FKBP12 transcript occur in the corpus striatum, with comparable levels evident in the substantia nigra zona compacta (31Dawson T.M Steiner J.P Lyons W.E Fotuhi M Blue M Snyder S.H The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain relationship to calcineurin.Neuroscience. 1994; 62: 569-580Crossref PubMed Scopus (124) Google Scholar). Lesioning studies indicate that these localizations reflect a very high FKBP12 content in the descending striatonigral pathway. High levels of FKBP12 are found in the hippocampus, with an enrichment in the CA1 layer. In the cerebral cortex, FKBP12 is most prominent in superficial layers, while in the cerebellum it is present in all cellular layers, with the highest density in the Purkinje cells. Parallel studies of calcineurin mRNA and protein densities reveal very similar localizations throughout the brain, implying a physiological link between calcineurin and FKBP12. Cyclophilin localizations also resemble calcineurin. Both are highly concentrated in cerebellar g