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Intramembrane Proteolysis of Astrotactins*

Open AccessPublished:January 18, 2017DOI:https://doi.org/10.1074/jbc.M116.768077
      Astrotactins are vertebrate-specific membrane proteins implicated in neuron-glia interactions during central nervous system development and in hair follicle polarity during skin development. By studying epitope-tagged derivatives of mouse astrotactin-2 (Astn2) produced in transfected cells, we determined that the amino and carboxyl termini reside in the extracellular space and are initially linked by two transmembrane segments and a single cytoplasmic domain. We further show that Astn2 undergoes proteolytic cleavage in the second transmembrane domain (TM2) and that a disulfide bond holds the resulting two fragments together. Recombinant Astn1 also undergoes TM2 cleavage, as does Astn2 isolated from mouse cerebellum. Astn2 intramembrane proteolysis is insensitive to replacement of TM2 by the transmembrane domain of CD74 or by 21 alanines. However, replacement of TM2 by the transmembrane domain of CD4, the asialoglycoprotein receptor, or the transferrin receptor eliminates intramembrane proteolysis, as does leucine substitution of residues that overlap or are immediately upstream of the cleavage site. Replacement of the transmembrane domain of CD74 or the asialoglycoprotein receptor with Astn2 TM2 leads to the appearance of a carboxyl-terminal fragment consistent with intramembrane proteolysis. These experiments define a highly unusual transmembrane topology for the astrotactins, reveal intramembrane proteolysis as a feature of astrotactin maturation, and constrain the substrate sequences that are permissive for cleavage of one type 2 transmembrane segment.

      Introduction

      Intramembrane proteolysis is a relatively unusual mode of polypeptide cleavage in which an integral membrane protease recognizes and cleaves a membrane-embedded substrate. At present, there are four known families of intramembrane proteases, and each uses a distinct catalytic mechanism: rhomboid family members are serine proteases, site 2 protease family members are metalloproteases, presenilin/γ-secretase/signal peptide peptidase (SPP)
      The abbreviations used are: SPP
      signal peptide peptidase
      BME
      β-mercaptoethanol
      IC
      intracellular
      SP
      signal peptide
      SPPL
      signal peptide peptidase-like
      SREBP
      sterol regulatory element binding proteins
      TM
      transmembrane
      RIPA
      radioimmune precipitation assay.
      family members are aspartyl proteases, and Rce1 (Ras and a-factor converting enzyme 1) is a glutamyl protease (
      • Golde T.E.
      • Wolfe M.S.
      • Greenbaum D.C.
      Signal peptide peptidases: a family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins.
      • Chen G.
      • Zhang X.
      New insights into S2P signaling cascades: regulation, variation, and conservation.
      ,
      • Urban S.
      • Dickey S.W.
      The rhomboid protease family: a decade of progress on function and mechanism.
      ,
      • Jurisch-Yaksi N.
      • Sannerud R.
      • Annaert W.
      A fast growing spectrum of biological functions of γ-secretase in development and disease.
      • Manolaridis I.
      • Kulkarni K.
      • Dodd R.B.
      • Ogasawara S.
      • Zhang Z.
      • Bineva G.
      • O'Reilly N.
      • Hanrahan S.J.
      • Thompson A.J.
      • Cronin N.
      • Iwata S.
      • Barford D.
      Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1.
      ). Intramembrane proteases are found in all kingdoms of life. In several well studied cases they play critical roles in development (cleavage and activation of epidermal growth factor-like ligands by rhomboid-1 in Drosophila and cleavage and release of the intracellular domain of Notch by γ-secretase), homeostasis (cleavage and activation of a tethered transcription factor that controls cholesterol synthesis and uptake by mammalian site-2 protease), and disease (processing of the amyloid-β peptide by γ-secretase).
      The SPP family is divided into two subfamilies based on the transmembrane topographies of enzyme and substrate (
      • Golde T.E.
      • Wolfe M.S.
      • Greenbaum D.C.
      Signal peptide peptidases: a family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins.
      ,
      • Voss M.
      • Schröder B.
      • Fluhrer R.
      Mechanism, specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases.
      ). The presenilin/γ-secretase subfamily cleaves transmembrane domains oriented with the carboxyl terminus facing the cytoplasm. By contrast, the SPP subfamily, which includes the SPP and SPP-like (SPPL) enzymes, has the opposite transmembrane topology relative to the presenilin/γ-secretase subfamily. SPP/SPPL subfamily members and the site 2 protease are the only known intramembrane proteases that cleave transmembrane domains that are oriented with the amino terminus facing the cytoplasm (type 2 orientation).
      Astn1 (astrotactin-1) and Astn2 (astrotactin-2) are homologous transmembrane proteins that have been implicated in neural development and in the response to CNS injury (
      • Fishell G.
      • Hatten M.E.
      Astrotactin provides a receptor system for CNS neuronal migration.
      ,
      • Zheng C.
      • Heintz N.
      • Hatten M.E.
      CNS gene encoding astrotactin, which supports neuronal migration along glial fibers.
      • Price M.
      • Lang M.G.
      • Frank A.T.
      • Goetting-Minesky M.P.
      • Patel S.P.
      • Silviera M.L.
      • Krady J.K.
      • Milner R.J.
      • Ewing A.G.
      • Day J.R.
      Seven cDNAs enriched following hippocampal lesion: possible roles in neuronal responses to injury.
      ). Astn1 is expressed widely in the CNS, whereas Astn2 is predominantly expressed in the cerebellum (
      • Wilson P.M.
      • Fryer R.H.
      • Fang Y.
      • Hatten M.E.
      Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration.
      ). In mice, Astn1 has been implicated in neuronal migration along glial scaffolds during CNS development based on ex vivo and gene knock-out experiments (
      • Fishell G.
      • Hatten M.E.
      Astrotactin provides a receptor system for CNS neuronal migration.
      ,
      • Adams N.C.
      • Tomoda T.
      • Cooper M.
      • Dietz G.
      • Hatten M.E.
      Mice that lack astrotactin have slowed neuronal migration.
      ). In humans, copy number variations affecting ASTN2 have been found in individuals with neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, obsessive-compulsive disorder, and schizophrenia (
      • Vrijenhoek T.
      • Buizer-Voskamp J.E.
      • van der Stelt I.
      • Strengman E.
      • Sabatti C.
      • Geurts van Kessel A.
      • Brunner H.G.
      • Ophoff R.A.
      • Veltman J.A.
      Genetic Risk and Outcome in Psychosis (GROUP) Consortium
      Recurrent CNVs disrupt three candidate genes in schizophrenia patients.
      ,
      • Glessner J.T.
      • Wang K.
      • Cai G.
      • Korvatska O.
      • Kim C.E.
      • Wood S.
      • Zhang H.
      • Estes A.
      • Brune C.W.
      • Bradfield J.P.
      • Imielinski M.
      • Frackelton E.C.
      • Reichert J.
      • Crawford E.L.
      • Munson J.
      • et al.
      Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.
      • Lionel A.C.
      • Tammimies K.
      • Vaags A.K.
      • Rosenfeld J.A.
      • Ahn J.W.
      • Merico D.
      • Noor A.
      • Runke C.K.
      • Pillalamarri V.K.
      • Carter M.T.
      • Gazzellone M.J.
      • Thiruvahindrapuram B.
      • Fagerberg C.
      • Laulund L.W.
      • Pellecchia G.
      • et al.
      Disruption of the ASTN2/TRIM32 locus at 9q33.1 is a risk factor in males for autism spectrum disorders, ADHD and other neurodevelopmental phenotypes.
      ). Astrotactins are also widely expressed in non-CNS tissues, and recent mouse genetic experiments have demonstrated a role for Astn2 in biasing the orientation of hair follicles in the context of impaired planar polarity signaling (
      • Chang H.
      • Cahill H.
      • Smallwood P.M.
      • Wang Y.
      • Nathans J.
      Identification of astrotactin2 as a genetic modifier that regulates the global orientation of mammalian hair follicles.
      ). In particular, both spontaneous and genetically engineered deletions of Astn2 exon5 produced a recessive genetic modifier of the hair polarity phenotype associated with homozygous knock-out of the planar cell polarity gene Frizzled6.
      Although the biochemical basis of astrotactin function is unknown, the three-dimensional structure of part of the carboxyl-terminal ectodomain of human ASTN2 has recently been determined, and this structure reveals a perforin-like domain, an EGF-like domain, a fibronectin type III domain, and an annexin-like domain (
      • Ni T.
      • Harlos K.
      • Gilbert R.
      Structure of astrotactin-2: a conserved vertebrate-specific and perforin-like membrane protein involved in neuronal development.
      ). Perforin domains are found in variety of membrane pore-forming proteins, but the structure of the ASTN2 perforin-like domain suggests that it is unlikely to form pores.
      In the present work, we have defined the transmembrane topology of mouse Astn2 and shown that this protein undergoes a single intramembrane proteolytic cleavage in the second of two transmembrane segments. The two fragments remain associated via a disulfide bond between a pair of cysteines very close to the amino terminus of each fragment. Intramembrane cleavage is likely mediated by a member of the SPP/SPPL family. The presence of extracellular amino and carboxyl termini together with intramembrane cleavage makes the structure and maturation of astrotactins highly unusual.

      Discussion

      The experiments reported here establish an unusual transmembrane topology for Astn2, in which both the amino and carboxyl termini reside within the extracellular space (or, equivalently, the lumen of an internal membrane system) with a large connecting loop residing within the cytoplasm. Our experiments further identify intramembrane proteolytic cleavage in TM2 and a single disulfide bond linking the amino termini of the two resulting fragments. Proteolysis within TM2 occurs efficiently and is insensitive to conversion of some or all of the TM2 amino acids to alanine or to swapping of CD74 TM sequences for Astn2 TM2 sequences. In light of the high degree of sequence conservation between Astn1 and Astn2, their nearly identical hydropathy profiles, the Edman sequencing data showing that Astn1 TM2 is cleaved at a site that corresponds to the TM2 cleavage site in Astn2, and the virtually identical BME-dependent mobility shifts exhibited by Astn1 and Astn2, it is highly likely that Astn1 adopts the same transmembrane topology and covalent structure as defined here for Astn2.

      Implications for Astrotactin Function

      Astn2 gene deletions that encompass exon 5 modify the Frizzled6−/− hair orientation phenotype, and sequences from multiple Astn1 and Astn2 cDNA clones and from Astn2 RT-PCR products show that some mature astrotactin transcripts lack exon 4 (
      • Chang H.
      • Cahill H.
      • Smallwood P.M.
      • Wang Y.
      • Nathans J.
      Identification of astrotactin2 as a genetic modifier that regulates the global orientation of mammalian hair follicles.
      ). The present work shows that the amino acids encoded by exons 4 and 5 are part of the intracellular loop connecting TM1 and TM2 and that the absence of either or both exons (creating in-frame deletions in each case) has no effect on the proteolytic processing or yield of the resulting Astn2 variants in transfected cells. These data suggest that Astn2 variants lacking amino acids encoded by exon 4 and/or exon 5 may exhibit protein activity. Future genetic experiments, including the construction of a definitive Astn2 null allele, should be able to determine whether the genetic modifier activity of Astn2 exon 5 deletion reflects an alteration of Astn2 function or a complete loss of Astn2 function.
      The low electrophoretic mobility of Astn2 from mouse cerebellum in the absence of BME strongly implies the existence of a disulfide-linked partner. Identification of the putative partner could provide significant insights into Astn2 biogenesis and function.

      Comparison with Proteolysis of Other Transmembrane Proteins

      Proteolysis of transmembrane proteins can be either regulated or constitutive. An example of the former is the cholesterol-dependent cleavage of SREBPs by the site 1 protease and its partner, SCAP (SREBP cleavage-activating protein) (
      • Brown M.S.
      • Goldstein J.L.
      A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.
      ). Examples of constitutive proteolysis in which the fragments remain associated, as they do for Astn2, include the cleavage of the insulin receptor, Lrp1 (low density lipoprotein-related receptor 1), and multiple members of the cell adhesion G-protein coupled receptor family (
      • Hedo J.A.
      • Kahn C.R.
      • Hayashi M.
      • Yamada K.M.
      • Kasuga M.
      Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits.
      • Herz J.
      • Kowal R.C.
      • Goldstein J.L.
      • Brown M.S.
      Proteolytic processing of the 600 kd low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment.
      ,
      • Alarcón C.
      • Cheatham B.
      • Lincoln B.
      • Kahn C.R.
      • Siddle K.
      • Rhodes C.J.
      A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor.
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). In each of these examples of constitutive proteolysis, cleavage occurs in the extracellular domain. For these proteins, and possibly for the astrotactins, proteolysis may facilitate or render irreversible a transition from an inactive to an active conformation. The high efficiency of astrotactin TM2 cleavage, together with the absence of any evidence for additional cleavage events beyond signal peptide removal, suggests that TM2 cleavage represents a constitutive step in astrotactin maturation, similar to the extracellular cleavage of insulin receptor or Lrp1.
      Intramembrane cleavage is often dependent on or stimulated by an initial proteolytic cleavage outside of the membrane. It is also typically associated with the dissociation of the soluble and membrane-embedded proteolytic products. For example, the basic helix-loop-helix DNA-binding transcription factor domain of SREBP is released by intramembrane cleavage, which is catalyzed by site 2 protease only after an initial cleavage by site 1 protease of an extramembrane loop in SREBP (
      • Brown M.S.
      • Goldstein J.L.
      A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.
      ). Similarly, the carboxyl-terminal cytoplasmic domain of Notch is released by intramembrane cleavage, which is catalyzed by γ-secretase after Notch-Jagged or Notch-Delta binding and subsequent extracellular domain cleavage by ADAM10 (A disintegrin and metalloprotease 10) (
      • van Tetering G.
      • van Diest P.
      • Verlaan I.
      • van der Wall E.
      • Kopan R.
      • Vooijs M.
      Metalloprotease ADAM10 is required for Notch1 site 2 cleavage.
      ,
      • Weber S.
      • Niessen M.T.
      • Prox J.
      • Lüllmann-Rauch R.
      • Schmitz A.
      • Schwanbeck R.
      • Blobel C.P.
      • Jorissen E.
      • de Strooper B.
      • Niessen C.M.
      • Saftig P.
      The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling.
      • Weber S.
      • Saftig P.
      Ectodomain shedding and ADAMs in development.
      ). By contrast, intramembrane cleavage of astrotactins does not appear to require a second proteolytic cleavage, nor does it lead to dissociation of the two proteolytic products.
      At present, the only known intramembrane proteases that cleave substrates with the type 2 topology of astrotactin TM2 are the site 2 protease and the SPP and SPPL peptidases (
      • Golde T.E.
      • Wolfe M.S.
      • Greenbaum D.C.
      Signal peptide peptidases: a family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins.
      ,
      • Voss M.
      • Schröder B.
      • Fluhrer R.
      Mechanism, specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases.
      ). Members of this family are highly conserved and are found in fungi, protozoa, plants, and animals (
      • Voss M.
      • Schröder B.
      • Fluhrer R.
      Mechanism, specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases.
      ). Studies of the substrate specificity of SPP and SPPL family members show that SPPLa and SPPL2b strongly favor substrates that have been subjected to ectodomain shedding, and SPP cleaves its signal peptide substrate following its release from the nascent polypeptide by signal peptidase (
      • Voss M.
      • Schröder B.
      • Fluhrer R.
      Mechanism, specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases.
      ,
      • Martin L.
      • Fluhrer R.
      • Haass C.
      Substrate requirements for SPPL2b-dependent regulated intramembrane proteolysis.
      ,
      • Zahn C.
      • Kaup M.
      • Fluhrer R.
      • Fuchs H.
      The transferrin receptor-1 membrane stub undergoes intramembrane proteolysis by signal peptide peptidase-like 2b.
      ,
      • Kirkin V.
      • Cahuzac N.
      • Guardiola-Serrano F.
      • Huault S.
      • Lückerath K.
      • Friedmann E.
      • Novac N.
      • Wels W.S.
      • Martoglio B.
      • Hueber A.O.
      • Zörnig M.
      The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells.
      ). However, SPPL3 makes an exception to this pattern by cleaving the first of two TM segments in the foamy virus envelope protein precursor in the absence of any other cleavage events (
      • Voss M.
      • Fukumori A.
      • Kuhn P.H.
      • Künzel U.
      • Klier B.
      • Grammer G.
      • Haug-Kröper M.
      • Kremmer E.
      • Lichtenthaler S.F.
      • Steiner H.
      • Schröder B.
      • Haass C.
      • Fluhrer R.
      Foamy virus envelope protein is a substrate for signal peptide peptidase-like 3 (SPPL3).
      ). SPPL3 also cleaves the membrane anchoring segments of a wide variety of glycan-modifying enzymes in the Golgi apparatus (
      • Voss M.
      • Künzel U.
      • Higel F.
      • Kuhn P.H.
      • Colombo A.
      • Fukumori A.
      • Haug-Kröper M.
      • Klier B.
      • Grammer G.
      • Seidl A.
      • Schröder B.
      • Obst R.
      • Steiner H.
      • Lichtenthaler S.F.
      • Haass C.
      • et al.
      Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation.
      ,
      • Kuhn P.H.
      • Voss M.
      • Haug-Kröper M.
      • Schröder B.
      • Schepers U.
      • Bräse S.
      • Haass C.
      • Lichtenthaler S.F.
      • Fluhrer R.
      Secretome analysis identifies novel signal peptide peptidase-like 3 (Sppl3) substrates and reveals a role of Sppl3 in multiple Golgi glycosylation pathways.
      ). Finally, recent studies of sequence determinants of transmembrane cleavage of CD74 by SPPL2a indicate that substrate specificity is determined by the combined effect of multiple residues in the CD74 transmembrane and juxtamembrane regions (
      • Hüttl S.
      • Helfrich F.
      • Mentrup T.
      • Held S.
      • Fukumori A.
      • Steiner H.
      • Saftig P.
      • Fluhrer R.
      • Schröder B.
      Substrate determinants of signal peptide peptidase-like 2a (SPPL2a)-mediated intramembrane proteolysis of the invariant chain CD74.
      ). Although the identity of the protease that cleaves the astrotactins remains to be determined, the observation of highly efficient astrotactin cleavage suggests the possibility that this type of transmembrane cleavage may be more widespread than is currently appreciated.

      Author Contributions

      H. C. and J. N. designed experiments. P. M. S. constructed many of the plasmids. J. W. produced anti-Astn2 antisera. H. C. conducted the experiments. H. C. and J. N. wrote the paper.

      Acknowledgments

      We thank Jodie Franklin of the Johns Hopkins Synthesis and Sequencing Facility for performing the amino-terminal Edman degradation, Amir Rattner for assistance with confocal microscopy, Sin Urban for advice on intramembrane proteolysis, Tao Ni and Robert Gilbert for advice on astrotactin structure, and Amir Rattner and Sin Urban for helpful comments on the manuscript.

      References

        • Golde T.E.
        • Wolfe M.S.
        • Greenbaum D.C.
        Signal peptide peptidases: a family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins.
        Semin. Cell Dev. Biol. 2009; 20: 225-230
        • Chen G.
        • Zhang X.
        New insights into S2P signaling cascades: regulation, variation, and conservation.
        Protein Sci. 2010; 19: 2015-2030
        • Urban S.
        • Dickey S.W.
        The rhomboid protease family: a decade of progress on function and mechanism.
        Genome Biol. 2011; 12: 231
        • Jurisch-Yaksi N.
        • Sannerud R.
        • Annaert W.
        A fast growing spectrum of biological functions of γ-secretase in development and disease.
        Biochim. Biophys. Acta. 2013; 1828: 2815-2827
        • Manolaridis I.
        • Kulkarni K.
        • Dodd R.B.
        • Ogasawara S.
        • Zhang Z.
        • Bineva G.
        • O'Reilly N.
        • Hanrahan S.J.
        • Thompson A.J.
        • Cronin N.
        • Iwata S.
        • Barford D.
        Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1.
        Nature. 2013; 504: 301-305
        • Voss M.
        • Schröder B.
        • Fluhrer R.
        Mechanism, specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases.
        Biochim. Biophys. Acta. 2013; 1828: 2828-2839
        • Fishell G.
        • Hatten M.E.
        Astrotactin provides a receptor system for CNS neuronal migration.
        Development. 1991; 113: 755-765
        • Zheng C.
        • Heintz N.
        • Hatten M.E.
        CNS gene encoding astrotactin, which supports neuronal migration along glial fibers.
        Science. 1996; 272: 417-419
        • Price M.
        • Lang M.G.
        • Frank A.T.
        • Goetting-Minesky M.P.
        • Patel S.P.
        • Silviera M.L.
        • Krady J.K.
        • Milner R.J.
        • Ewing A.G.
        • Day J.R.
        Seven cDNAs enriched following hippocampal lesion: possible roles in neuronal responses to injury.
        Brain Res. Mol. Brain Res. 2003; 117: 58-67
        • Wilson P.M.
        • Fryer R.H.
        • Fang Y.
        • Hatten M.E.
        Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration.
        J. Neurosci. 2010; 30: 8529-8540
        • Adams N.C.
        • Tomoda T.
        • Cooper M.
        • Dietz G.
        • Hatten M.E.
        Mice that lack astrotactin have slowed neuronal migration.
        Development. 2002; 129: 965-972
        • Vrijenhoek T.
        • Buizer-Voskamp J.E.
        • van der Stelt I.
        • Strengman E.
        • Sabatti C.
        • Geurts van Kessel A.
        • Brunner H.G.
        • Ophoff R.A.
        • Veltman J.A.
        • Genetic Risk and Outcome in Psychosis (GROUP) Consortium
        Recurrent CNVs disrupt three candidate genes in schizophrenia patients.
        Am. J. Hum. Genet. 2008; 83: 504-510
        • Glessner J.T.
        • Wang K.
        • Cai G.
        • Korvatska O.
        • Kim C.E.
        • Wood S.
        • Zhang H.
        • Estes A.
        • Brune C.W.
        • Bradfield J.P.
        • Imielinski M.
        • Frackelton E.C.
        • Reichert J.
        • Crawford E.L.
        • Munson J.
        • et al.
        Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.
        Nature. 2009; 459: 569-573
        • Lionel A.C.
        • Tammimies K.
        • Vaags A.K.
        • Rosenfeld J.A.
        • Ahn J.W.
        • Merico D.
        • Noor A.
        • Runke C.K.
        • Pillalamarri V.K.
        • Carter M.T.
        • Gazzellone M.J.
        • Thiruvahindrapuram B.
        • Fagerberg C.
        • Laulund L.W.
        • Pellecchia G.
        • et al.
        Disruption of the ASTN2/TRIM32 locus at 9q33.1 is a risk factor in males for autism spectrum disorders, ADHD and other neurodevelopmental phenotypes.
        Hum. Mol. Genet. 2014; 23: 2752-2768
        • Chang H.
        • Cahill H.
        • Smallwood P.M.
        • Wang Y.
        • Nathans J.
        Identification of astrotactin2 as a genetic modifier that regulates the global orientation of mammalian hair follicles.
        PLoS Genet. 2015; 11e1005532
        • Ni T.
        • Harlos K.
        • Gilbert R.
        Structure of astrotactin-2: a conserved vertebrate-specific and perforin-like membrane protein involved in neuronal development.
        Open Biol. 2016; 6160053
        • Sun H.
        • Molday R.S.
        • Nathans J.
        Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease.
        J. Biol. Chem. 1999; 274: 8269-8281
        • Lemberg M.K.
        • Martoglio B.
        Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis.
        Mol. Cell. 2002; 10: 735-744
        • Martin L.
        • Fluhrer R.
        • Haass C.
        Substrate requirements for SPPL2b-dependent regulated intramembrane proteolysis.
        J. Biol. Chem. 2009; 284: 5662-5670
        • Fluhrer R.
        • Martin L.
        • Klier B.
        • Haug-Kröper M.
        • Grammer G.
        • Nuscher B.
        • Haass C.
        The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b).
        J. Biol. Chem. 2012; 287: 5156-5163
        • Parnes J.R.
        • Hunkapiller T.
        L3T4 and the immunoglobulin gene superfamily: new relationships between the immune system and the nervous system.
        Immunol. Rev. 1987; 100: 109-127
        • Schneider C.
        • Owen M.J.
        • Banville D.
        • Williams J.G.
        Primary structure of human transferrin receptor deduced from the mRNA sequence.
        Nature. 1984; 311: 675-678
        • Zerial M.
        • Melancon P.
        • Schneider C.
        • Garoff H.
        The transmembrane segment of the human transferrin receptor functions as a signal peptide.
        EMBO J. 1986; 5: 1543-1550
        • Drickamer K.
        Membrane receptors that mediate glycoprotein endocytosis: structure and biosynthesis.
        Kidney Int. Suppl. 1987; 23: S167-S183
        • Stumptner-Cuvelette P.
        • Benaroch P.
        Multiple roles of the invariant chain in MHC class II function.
        Biochim. Biophys. Acta. 2002; 1542: 1-13
        • Beisner D.R.
        • Langerak P.
        • Parker A.E.
        • Dahlberg C.
        • Otero F.J.
        • Sutton S.E.
        • Poirot L.
        • Barnes W.
        • Young M.A.
        • Niessen S.
        • Wiltshire T.
        • Bodendorf U.
        • Martoglio B.
        • Cravatt B.
        • Cooke M.P.
        The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain.
        J. Exp. Med. 2013; 210: 23-30
        • Bergmann H.
        • Yabas M.
        • Short A.
        • Miosge L.
        • Barthel N.
        • Teh C.E.
        • Roots C.M.
        • Bull K.R.
        • Jeelall Y.
        • Horikawa K.
        • Whittle B.
        • Balakishnan B.
        • Sjollema G.
        • Bertram E.M.
        • Mackay F.
        • et al.
        B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.
        J. Exp. Med. 2013; 210: 31-40
        • Schneppenheim J.
        • Dressel R.
        • Hüttl S.
        • Lüllmann-Rauch R.
        • Engelke M.
        • Dittmann K.
        • Wienands J.
        • Eskelinen E.L.
        • Hermans-Borgmeyer I.
        • Fluhrer R.
        • Saftig P.
        • Schröder B.
        The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain.
        J. Exp. Med. 2013; 210: 41-58
        • Zahn C.
        • Kaup M.
        • Fluhrer R.
        • Fuchs H.
        The transferrin receptor-1 membrane stub undergoes intramembrane proteolysis by signal peptide peptidase-like 2b.
        FEBS J. 2013; 280: 1653-1663
        • Brown M.S.
        • Goldstein J.L.
        A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.
        Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11041-11048
        • Hedo J.A.
        • Kahn C.R.
        • Hayashi M.
        • Yamada K.M.
        • Kasuga M.
        Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits.
        J. Biol. Chem. 1983; 258: 10020-10026
        • Herz J.
        • Kowal R.C.
        • Goldstein J.L.
        • Brown M.S.
        Proteolytic processing of the 600 kd low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment.
        EMBO J. 1990; 9: 1769-1776
        • Alarcón C.
        • Cheatham B.
        • Lincoln B.
        • Kahn C.R.
        • Siddle K.
        • Rhodes C.J.
        A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor.
        Biochem. J. 1994; 301: 257-265
        • Araç D.
        • Boucard A.A.
        • Bolliger M.F.
        • Nguyen J.
        • Soltis S.M.
        • Südhof T.C.
        • Brunger A.T.
        A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
        EMBO J. 2012; 31: 1364-1378
        • van Tetering G.
        • van Diest P.
        • Verlaan I.
        • van der Wall E.
        • Kopan R.
        • Vooijs M.
        Metalloprotease ADAM10 is required for Notch1 site 2 cleavage.
        J. Biol. Chem. 2009; 284: 31018-31027
        • Weber S.
        • Niessen M.T.
        • Prox J.
        • Lüllmann-Rauch R.
        • Schmitz A.
        • Schwanbeck R.
        • Blobel C.P.
        • Jorissen E.
        • de Strooper B.
        • Niessen C.M.
        • Saftig P.
        The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling.
        Development. 2011; 138: 495-505
        • Weber S.
        • Saftig P.
        Ectodomain shedding and ADAMs in development.
        Development. 2012; 139: 3693-3709
        • Kirkin V.
        • Cahuzac N.
        • Guardiola-Serrano F.
        • Huault S.
        • Lückerath K.
        • Friedmann E.
        • Novac N.
        • Wels W.S.
        • Martoglio B.
        • Hueber A.O.
        • Zörnig M.
        The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells.
        Cell Death Differ. 2007; 14: 1678-1687
        • Voss M.
        • Fukumori A.
        • Kuhn P.H.
        • Künzel U.
        • Klier B.
        • Grammer G.
        • Haug-Kröper M.
        • Kremmer E.
        • Lichtenthaler S.F.
        • Steiner H.
        • Schröder B.
        • Haass C.
        • Fluhrer R.
        Foamy virus envelope protein is a substrate for signal peptide peptidase-like 3 (SPPL3).
        J. Biol. Chem. 2012; 287: 43401-43409
        • Voss M.
        • Künzel U.
        • Higel F.
        • Kuhn P.H.
        • Colombo A.
        • Fukumori A.
        • Haug-Kröper M.
        • Klier B.
        • Grammer G.
        • Seidl A.
        • Schröder B.
        • Obst R.
        • Steiner H.
        • Lichtenthaler S.F.
        • Haass C.
        • et al.
        Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation.
        EMBO J. 2014; 33: 2890-2905
        • Kuhn P.H.
        • Voss M.
        • Haug-Kröper M.
        • Schröder B.
        • Schepers U.
        • Bräse S.
        • Haass C.
        • Lichtenthaler S.F.
        • Fluhrer R.
        Secretome analysis identifies novel signal peptide peptidase-like 3 (Sppl3) substrates and reveals a role of Sppl3 in multiple Golgi glycosylation pathways.
        Mol. Cell. Proteomics. 2015; 14: 1584-1598
        • Hüttl S.
        • Helfrich F.
        • Mentrup T.
        • Held S.
        • Fukumori A.
        • Steiner H.
        • Saftig P.
        • Fluhrer R.
        • Schröder B.
        Substrate determinants of signal peptide peptidase-like 2a (SPPL2a)-mediated intramembrane proteolysis of the invariant chain CD74.
        Biochem. J. 2016; 473: 1405-1422
        • Illing M.
        • Molday L.L.
        • Molday R.S.
        The 220-kDa rim protein of retinal rod outer segments is a member of the ABC transporter superfamily.
        J. Biol. Chem. 1997; 272: 10303-10310
        • Nathans J.
        Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin.
        Biochemistry. 1990; 29: 9746-9752