Alternative Splicing, Expression, and Genomic Structure of the 3′ Region of the Gene Encoding the Sarcolemmal-associated Proteins (SLAPs) Defines a Novel Class of Coiled-coil Tail-anchored Membrane Proteins

The sarcolemmal associated proteins (SLAPs) are encoded by multiple mRNAs that are presumably generated by alternative splicing mechanisms. The amino acid sequence of the SLAP1 isoform exhibited 76% identity with TOPAP, a topographically graded antigen of the chick visual system. The regions of coiled-coil structure including an 11-heptad acidic amphipathic α-helical segment was conserved with a major divergence in sequence noted in the hydrophobic C termini predicted to be transmembrane domains in the two polypeptides. The genomic organization of the 3′ region of the SLAP gene indicated that SLAP1 and TOPAP are generated by alternative splicing mechanisms, which are conserved among mammalian and avian species. SLAP1/TOPAP were encoded by 11 exons distributed over a minimum of 35 kilobase pairs of continuous DNA; 9 of the exons were constitutively expressed, and 2 were alternatively spliced. The exons range in size from 60 to 321 base pairs, and the predicted functional domains within the polypeptides were encompassed by single exons. The introns vary from 0.2 to 10 kilobase pairs and conform to consensus dinucleotide splicing signals. Reverse transcriptase-polymerase chain reaction studies demonstrated that alternative exons (IV and X) of SLAP were expressed in a tissue-specific fashion and developmentally regulated. The alternatively spliced exon X, which encodes the putative transmembrane anchor in TOPAP, and a constitutively expressed exon XI, which encodes the putative transmembrane domain in SLAP, were found to target these polypeptides to membrane structures. The presence and conservation of termination codons in exons X and XI render expression of the two SLAP1/TOPAP transmembrane domains mutually exclusive. These data reveal that TOPAP and SLAP are alternatively spliced products of a single gene that encodes a unique class of tail-anchored membrane proteins. The sarcolemmal associated proteins (SLAPs) are encoded by multiple mRNAs that are presumably generated by alternative splicing mechanisms. The amino acid sequence of the SLAP1 isoform exhibited 76% identity with TOPAP, a topographically graded antigen of the chick visual system. The regions of coiled-coil structure including an 11-heptad acidic amphipathic α-helical segment was conserved with a major divergence in sequence noted in the hydrophobic C termini predicted to be transmembrane domains in the two polypeptides. The genomic organization of the 3′ region of the SLAP gene indicated that SLAP1 and TOPAP are generated by alternative splicing mechanisms, which are conserved among mammalian and avian species. SLAP1/TOPAP were encoded by 11 exons distributed over a minimum of 35 kilobase pairs of continuous DNA; 9 of the exons were constitutively expressed, and 2 were alternatively spliced. The exons range in size from 60 to 321 base pairs, and the predicted functional domains within the polypeptides were encompassed by single exons. The introns vary from 0.2 to 10 kilobase pairs and conform to consensus dinucleotide splicing signals. Reverse transcriptase-polymerase chain reaction studies demonstrated that alternative exons (IV and X) of SLAP were expressed in a tissue-specific fashion and developmentally regulated. The alternatively spliced exon X, which encodes the putative transmembrane anchor in TOPAP, and a constitutively expressed exon XI, which encodes the putative transmembrane domain in SLAP, were found to target these polypeptides to membrane structures. The presence and conservation of termination codons in exons X and XI render expression of the two SLAP1/TOPAP transmembrane domains mutually exclusive. These data reveal that TOPAP and SLAP are alternatively spliced products of a single gene that encodes a unique class of tail-anchored membrane proteins. sarcolemmal-associatedprotein topographically graded antigen of the chick visual system reverse transcriptase-polymerase chain reaction transmembrane base pairs nucleotide(s) The coiled-coil membrane proteins such as syntaxin, synaptobrevin, and epimorphin are believed to play diverse roles in cell function including membrane fusion, vesicle transport, and neurotransmitter release (1Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1020) Google Scholar, 2Rothman J.E. Scheller R.H. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 3Bennett M.K. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2559-2563Crossref PubMed Scopus (547) Google Scholar, 4Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-419Abstract Full Text PDF PubMed Scopus (1578) Google Scholar). These polypeptides belong to a growing family of membrane proteins that lack a signal sequence and are anchored in the membrane by a hydrophobic segment at the C terminus (5Kutay U. Hartmann E. Rapoport T.A. Trends. Cell Biol. 1993; 3: 72-75Abstract Full Text PDF PubMed Scopus (267) Google Scholar). These tail-anchored proteins can be found at the plasma membrane as well as on intracellular membrane compartments, and evidence suggests that gene families as well as alternative splicing mechanisms may generate products with distinct hydrophobic C-terminal peptides that determine subcellular localizations and hence function (6Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1071) Google Scholar, 7Spring J.M. Kato M. Bernfield M. Trends. Biochem. Sci. 1993; 18: 124-125Abstract Full Text PDF PubMed Scopus (24) Google Scholar). The membrane insertion of these polypeptides is believed to be achieved by unique mechanisms that do not require the signal recognition particle (8Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Crossref PubMed Scopus (260) Google Scholar). An important feature in these polypeptides is an extended coiled-coil structure, believed to be important for their intermolecular interactions (9Zhang P. Chen Y.A. Tam D. Chung D. Scheller R.H. Miljanich G.P. Biochemistry. 1997; 36: 4317-4326Crossref PubMed Scopus (39) Google Scholar, 10Chapman E.R. An S. Barton H. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google Scholar, 11Sheng Z.H. Rettig J. Cook T. Catterall W.A. Nature. 1996; 379: 451-454Crossref PubMed Scopus (310) Google Scholar). We have previously defined the cloning of a series of cDNAs that encode polypeptides, which were found to be associated with cardiac membranes, referred to as SLAPs1 (for sarcolemmal-associated proteins (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar)). Three different SLAP transcripts expressed in cardiac tissue were found to contain unique 5′ and common 3′ sequences that encode three SLAP polypeptides (37-kDa SLAP1, 46-kDa SLAP2, and 74-kDa SLAP3). The most noticeable structural features of SLAPs include their predicted ability to form an extended coiled coil over most of their length and a putative transmembrane domain at the extreme C terminus, although there was no consensus signal sequence (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Our previous studies indicate that SLAPs are integral membrane proteins that localize to the cell membrane as well as intracellular membranes, but it is not known whether the putative transmembrane domain found in the SLAPs is responsible for their membrane integration (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Although we do not know the physiological role of the SLAPs, the proteins share common core features with the tail-anchored membrane proteins and hence may play important roles in membrane biology. In this regard, homology searches of the GenBankTM data base revealed that the SLAP1 isoform shares high nucleotide and amino acid sequence identity with TOPAP, a topographically graded antigen of the chick visual system implicated in the neuronal guidance and development of the chick retinotectal map (13Savitt J.M. Trisler D. Hilt D.C. Neuron. 1995; 14: 253-261Abstract Full Text PDF PubMed Scopus (37) Google Scholar). The molecular properties of TOPAP reveal that it is a 35-kDa coiled-coil protein with a putative transmembrane domain at the C terminus, which is believed to be localized at the cell membrane. There was strong identity (76% at the amino acid level) between avian TOPAP and the 37-kDa SLAP1 isoform over most of the coding region, with the differences noted in the predicted transmembrane domains in the two polypeptides. This finding led us to hypothesize that avian TOPAP and mammalian SLAP may be derived by alternative splicing mechanisms from a common gene. We therefore undertook studies to determine the genomic organization of the 3′ region of the SLAP gene, which revealed that mammalian SLAP1 and avian TOPAP are generated by alternative splicing mechanisms that are highly conserved among mammalian and avian species. In addition, we report that the alternatively spliced products were expressed in a developmental and tissue-specific manner and the putative transmembrane domains were responsible for their membrane integration. The screening of the rabbit genomic λ DashII library (Stratagene) with a radiolabeled restriction fragment of SLAP3 cDNA (U21157; 324 nt AvaI-HindIII, 1607–1931) led to the isolation of three positive clones (R-2, R-13, R-14). A full-length SLAP3 cDNA probe, SalI-EcoRI (nt 1–3017), was also used to screen a mouse genomic λ DashII library, and two additional clones (M-2 and M-3) were isolated. The genomic clones were digested with EcoRI, BamHI, and KpnI, resolved on agarose gels, and blotted onto MCI nylon membranes. The membranes were hybridized at 65 °C for 16 h, withAvaI-HindIII and SalI-EcoRI cDNA radiolabeled probes in 10% polyethylene glycol, 7% SDS and 2.5× saline/sodium phosphate/EDTA. The membranes were then washed at 60 °C for 30 min and using intensifying screens were exposed to Kodak BioMax MR film at −70 °C for 16 to 24 h. The positive restriction fragments of the genomic λ clones were subcloned into pBlueScript KS (Stratagene) and sequenced. Mouse spleen and human cardiac cDNA libraries constructed in λZAP were screened with aSalI-EcoRI restriction fragment of rabbit SLAP3 cDNA encompassing the entire coding region of SLAP3 (U21157, nt 1–3017). Among the various positive clones obtained, mouse spleen clone 15.1 and human cardiac clone 12.3 were in vivo excised, and the nucleotide sequences were determined using AmpliTaqsequencing methodology (ABI) and analyzed with SeqAidII (University of Kansas), PROSITE, PSORT, and BLAST. Total RNA was prepared from dissected tissues using the TriPure methodology (Roche Molecular Biochemicals). For developmental expression, RNA was prepared from rat heart, brain, and muscle at fetal (day 18 of gestation), neonatal (day 4), and adult stages. First-strand cDNA synthesis reaction contained 1.0 μg of RNA, 200 units of SuperScript reverse transcriptase (Life Technologies Inc.), and random hexamers. 10 μl of the first-strand product was used as a template in PCR reactions (50 μl total) with a final concentration of 10 mm Tris-HCl, pH mm mm and nt nt nt and nt A single of was by at °C for by °C for °C for and °C for The was for min at The were resolved on a agarose and and the was by restriction of with restriction PCR products were subcloned and sequenced. of the 3′ of the SLAP was with nt and TOPAP, nt on the conserved sequences among mouse and human In a series of the fragments on agarose were with and with the system to determine the of the different size A SLAP expression in was generated as previously (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). SLAP of the putative domains by exons X and XI, was generated by PCR of SLAP with the and the PCR was restriction digested with and subcloned into the pBlueScript KS The SLAP cDNA domains was then with to the expression and as A SLAP an alternative domain encoded by exon X was constructed by of the exon with restriction and into the domains and as A a constitutively expressed putative domain encoded by exon XI was prepared by restriction of SLAP with and and in to the pBlueScript KS of the with the was to and as were with SLAP and using the to in and for with as previously (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). of was to and J. Cell Biol. PubMed Scopus Google and the proteins were analyzed by by with as previously (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). studies have three SLAP SLAP1, SLAP2, and encoded by a single gene to human SLAP contain common core C-terminal sequences that are highly with sequences to different human identity with the C-terminal sequence in rabbit SLAP, strong conservation and functional of this region of the the genomic organization of the SLAP gene encompassing the common core region, rabbit and mouse genomic λ DashII libraries were screened with SLAP cDNA restriction fragments used as probes (U21157, nt and A of positive genomic clones from rabbit (R-2, R-13, and mouse (M-2 and M-3) were and the coding regions and of the rabbit and were by restriction and The various fragments were subcloned and to determine of genomic λ clones revealed that the C-terminal common region of the SLAP (U21157, nt and the entire region of the SLAP1 The indicate that the SLAP1 isoform is of 11 exons over 35 kilobase pairs of genomic The exons range in size from 60 to 321 exons and were in SLAP1, are coding exons in and SLAP3 The of the SLAP1 isoform in exon to the consensus of this of a acid of as previously to be expressed in cardiac and contain the predicted coiled-coil exon in addition, a of XI encodes a of hydrophobic that a transmembrane anchor of and X were found to be alternatively spliced organization of the 3′ region of the SLAP gene. and mouse genomic λ DashII libraries were screened with two SLAP cDNA probes (U21157; nt and to positive genomic clones (R-2, R-13, and from rabbit and and from was restriction and to the in the rabbit and of genomic λ clones revealed the presence of 11 exons that encode the common C-terminal core of the three SLAP2, (U21157; nt of the encompassing the common region of SLAP conform to the consensus and 2 PubMed Scopus Google Scholar). In addition, the in mouse and rabbit were also determined to be conserved 2 The size of the SLAP introns range from 0.2 to kilobase of the introns with a and with an and class the coding sequence between the and of the to class 2 the and the of the and introns and 11 are class between introns and introns of the the are for exon as the alternative exons and X are on by class class the alternative splicing in a conservation of the of the molecular of SLAP, human cardiac and mouse spleen λZAP cDNA libraries were screened using a rabbit cDNA fragment (U21157, nt as a A of and positive clones were from the human and mouse a sequence the common SLAP regions were found to be highly although two of sequence divergence were with the previously rabbit SLAP1 sequence The nucleotide sequence revealed that mouse and human SLAP1 cDNAs contain sequence of and nt that are highly conserved between the two species. the of the sequence are in human and mouse and of rabbit SLAP The revealed that of the are in with the of SLAP splicing is conserved among mammalian and avian species. of RNA from was as indicated in the to to the 5′ and 3′ regions of divergence in the PCR fragments were and the sequenced. The amino acid sequence of the 5′ and 3′ regions of SLAP from rabbit heart, rat mouse human and that the alternative sequences are in with the SLAP1 sequence and highly conserved among A and the of insertion are also conserved among the various A a acid sequence that is highly conserved among species. a acid that a termination that also was conserved among analyzed to sequence that the rabbit sequence an additional that is in determine whether the sequences are also in rabbit SLAP, of RNA from rabbit was with and as well as and to SLAP the regions of cardiac tissue was as previous studies have that the is a of the different SLAP (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The in indicate that two distinct fragments were by of in the 5′ region and the 3′ region and of SLAP from RNA An additional fragment of was from RNA with the fragment from SLAP1 cDNA in the 5′ A fragment of was from RNA in to the fragment from SLAP1 cDNA in the 3′ of these a sequence revealed that the fragment contained the sequence in the 5′ region of SLAP, and the fragment contained the sequence at the 3′ region of The two rabbit cardiac were also generated by the of and sequences and as noted in the human and mouse SLAP that of the and sequences be generated by alternative splicing and are highly conserved regions among various species. A of the SLAP1 sequence indicated that it 76% identity with avian TOPAP a protein expressed in a in the (13Savitt J.M. Trisler D. Hilt D.C. Neuron. 1995; 14: 253-261Abstract Full Text PDF PubMed Scopus (37) Google Scholar). indicated that avian TOPAP also the and sequences in the SLAP1 determine whether the mammalian also these SLAP1 an of RNA from rat was with the 5′ and 3′ and two fragments were that contained the and the sequence The of the amino acid sequence of and chick SLAP1 are in A that the sequence encodes a acid sequence that is highly conserved among species. that the sequence the C-terminal acid a termination that is also conserved among analyzed The 3′ alternative sequence therefore encode an alternative hydrophobic C terminus in SLAP polypeptides. sequence that the rabbit sequence an additional which is in species. The of the at it is that the and sequences that were the alternatively spliced exons and X of the SLAP gene. In of the strong conservation of sequence and alternative splicing of exons and X in mammalian and avian studies were to determine the tissue-specific expression of these SLAP was to SLAP expression in various rabbit tissues with and to the 5′ alternative regions and and for the 3′ alternative of The of of tissue-specific expression of the 5′ region of SLAP reveal that of the tissues the two a molecular size fragment of which encode a with the alternative exon and a fragment of the alternative exon In muscle and the molecular size isoform the alternative exon to be the the spleen and the molecular size isoform the alternative exon In the expression of to be of the tissue-specific expression of the 3′ alternative exon also the using and which two products of and The of the indicate that of the tissues the two with and the alternative exon these are expressed at different the and and a expression of the molecular size isoform the alternative exon spleen and the molecular size isoform the alternative exon of the tissues to of the two alternatively spliced splicing a that tissue-specific and developmentally of gene determine whether the alternatively spliced of SLAPs were expressed in a developmentally from three distinct of rat development (day 18 of gestation), and adult was by with that the regions in SLAP and A of the 5′ alternative region of SLAP from various developmental with the and which in of two products of and with and exon of the by of the of at of development is indicated In the heart, the expression of the alternative exon is at of development to the with the alternative exon In brain, on the the expression of the is at the fetal with the this of expression is to reverse in the neonatal and adult The of expression of the in muscle was found to from to development to the of the 3′ alternative region of SLAP using and and RNA from various developmental in of and with and exon X, In the heart, the expression of the from at the neonatal to in the adult with the In brain, on the the expression of the is at the fetal and neonatal with the the expression of the in the adult was then the The expression of the was in neonatal and fetal muscle and by in adult Although SLAP and TOPAP have to be integral membrane proteins that lack a signal it to be defined whether the hydrophobic C termini their membrane integration. of SLAP the putative domains with were into and the by and subcellular of the with a of the and membrane in SLAP the putative SLAP these sequences exhibited a of the of these was and the various were by and with and SLAP was found to be in the membrane the and was in the therefore that hydrophobic sequences encoded in exons X and XI to the SLAP into membrane We have the genomic organization of the 3′ region of the SLAP gene the common C-terminal region of the various SLAP The common region (nt of SLAP3 cDNA over 35 kilobase pairs of continuous and of 11 exons. of the the consensus and rabbit and mouse SLAP gene are in the common region, of conservation of genomic structure and splicing mechanisms. In addition, the that SLAP isoform of alternative of the terminus, is by alternative splicing of a in two and X) of the 3′ region of the SLAP gene. The sequence of the alternatively spliced SLAP was found to be to TOPAP, a coiled-coil membrane protein to be in the development of the avian retinotectal avian TOPAP and mammalian SLAP1 are spliced and the conservation of the alternative splicing in and suggests that alternative exons may have functional roles that have by splicing of exon in an of a sequence a amino acid that is predicted to and by 11 the α-helical structure of SLAP1 (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, J.M. Trisler D. Hilt D.C. Neuron. 1995; 14: 253-261Abstract Full Text PDF PubMed Scopus (37) Google Scholar). splicing of exon also in the of a putative a found in the and which multiple in of cell and signal J.E. J. 1995; PubMed Scopus Google Scholar). This putative by alternative splicing is as the SLAP3 isoform putative for Although in and in vivo studies of this to be conservation among suggests a functional The alternative exon X, on the a highly hydrophobic segment a termination and the putative transmembrane domain found in splicing of exon X, a with a putative alternative membrane and subcellular studies of SLAP polypeptides exon X XI demonstrated that these sequences are for SLAP integration into membranes, the of these exons in of SLAP from membrane structures. mutually transmembrane domains encoded by exons X and XI, are responsible for SLAP to membrane of the alternatively spliced of SLAP revealed developmental in the expression of the alternative exons and X in tissues such as the In addition, expression of the alternative was found to be in a tissue-specific The developmental and tissue-specific splicing of the alternative exons is of a functional of the alternative SLAP to alternative and to alternative splicing generate to distinct SLAP Although the functional role of the SLAP to be it is that of the have localized to the cell membrane as well as to intracellular membranes, a role in membrane function (12Wigle J.T. Demchyshyn L. Pratt M.A.C. Staines W.A. Salih M. Tuana B.S. J. Biol. Chem. 1997; 272: 32384-32394Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, J.M. Trisler D. Hilt D.C. Neuron. 1995; 14: 253-261Abstract Full Text PDF PubMed Scopus (37) Google Scholar). In this regard, avian TOPAP, which data to be a SLAP to be localized at the cell to a role in neuronal guidance in the chick it is that the mammalian a In this regard, transcripts for SLAP1 and the TOPAP were found to be expressed in mammalian is from the that the SLAPs are to the membrane by the alternatively spliced C-terminal hydrophobic In this regard, the SLAPs belong to the family of tail-anchored coiled-coil membrane which lack a signal sequence and contain a C-terminal transmembrane of the tail-anchored family are in diverse and important including and of with plasma membrane and and intracellular vesicle that the family of proteins form intermolecular interactions their coiled-coil with protein of the vesicle membrane fusion, and membrane is a neuronal cell membrane protein that with (9Zhang P. Chen Y.A. Tam D. Chung D. Scheller R.H. Miljanich G.P. Biochemistry. 1997; 36: 4317-4326Crossref PubMed Scopus (39) Google E.R. An S. Barton H. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google and with a of vesicle In addition, also with the Z.H. Rettig J. Cook T. Catterall W.A. Nature. 1996; 379: 451-454Crossref PubMed Scopus (310) Google it to the of vesicle The between of is by α-helical domains that form intermolecular coiled-coil structures. is that 2 is a tail-anchored family known to contain alternative transmembrane domains generated by splicing mechanisms to that for the SLAP M.K. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2559-2563Crossref PubMed Scopus (547) Google Scholar). In of the that different C termini generated by alternative a function for the to the membrane is the SLAP by of their structural features and are to play roles in cell

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