Evolutionarily Conserved Multiple C2 Domain Proteins with Two Transmembrane Regions (MCTPs) and Unusual Ca2+ Binding Properties

C2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C2 domains. In both classes of proteins, C2 domains usually regulate the respective protein's function by forming Ca2+-dependent or Ca2+-independent phospholipid complexes. We now describe MCTPs (multiple C2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C2 domain proteins with unusual Ca2+-dependent properties. MCTPs are composed of a variable N-terminal sequence, three C2 domains, two transmembrane regions, and a short C-terminal sequence. The invertebrate organisms Caenorhabditis elegans and Drosophila melanogaster express a single MCTP gene, whereas vertebrates express two MCTP genes (MCTP1 and MCTP2) whose primary transcripts are extensively alternatively spliced. Most of the MCTP sequences, in particular the C2 domains, are highly conserved. All MCTP C2 domains except for the second C2 domain of MCTP2 include a perfect Ca2+/phospholipid-binding consensus sequence. To determine whether the C2 domains of MCTPs actually function as Ca2+/phospholipid-binding modules, we analyzed their Ca2+ and phospholipid binding properties. Surprisingly, we found that none of the three MCTP1 C2 domains interacted with negatively charged or neutral phospholipids in the presence or absence of Ca2+. However, Ca2+ titrations monitored via intrinsic tryptophan fluorescence revealed that all three C2 domains bound Ca2+ in the absence of phospholipids with a high apparent affinity (EC50 of ∼1.3-2.3 μm). Our data thus reveal that MCTPs are evolutionarily conserved C2 domain proteins that are unusual in that the C2 domains are anchored in the membrane by two closely spaced transmembrane regions and represent Ca2+-binding but not phospholipid-binding modules. C2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C2 domains. In both classes of proteins, C2 domains usually regulate the respective protein's function by forming Ca2+-dependent or Ca2+-independent phospholipid complexes. We now describe MCTPs (multiple C2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C2 domain proteins with unusual Ca2+-dependent properties. MCTPs are composed of a variable N-terminal sequence, three C2 domains, two transmembrane regions, and a short C-terminal sequence. The invertebrate organisms Caenorhabditis elegans and Drosophila melanogaster express a single MCTP gene, whereas vertebrates express two MCTP genes (MCTP1 and MCTP2) whose primary transcripts are extensively alternatively spliced. Most of the MCTP sequences, in particular the C2 domains, are highly conserved. All MCTP C2 domains except for the second C2 domain of MCTP2 include a perfect Ca2+/phospholipid-binding consensus sequence. To determine whether the C2 domains of MCTPs actually function as Ca2+/phospholipid-binding modules, we analyzed their Ca2+ and phospholipid binding properties. Surprisingly, we found that none of the three MCTP1 C2 domains interacted with negatively charged or neutral phospholipids in the presence or absence of Ca2+. However, Ca2+ titrations monitored via intrinsic tryptophan fluorescence revealed that all three C2 domains bound Ca2+ in the absence of phospholipids with a high apparent affinity (EC50 of ∼1.3-2.3 μm). Our data thus reveal that MCTPs are evolutionarily conserved C2 domain proteins that are unusual in that the C2 domains are anchored in the membrane by two closely spaced transmembrane regions and represent Ca2+-binding but not phospholipid-binding modules. The C2 domain is defined as a sequence motif in a comparison of the primary structures of different protein kinase C isoforms and named in an unbiased fashion as the “second constant sequence” of protein kinase C isozymes (1Coussens L. Parker P.J. Rhee L. Yang-Feng T.L. Chen E. Waterfield M.D. Francke U. Ullrich A. Science. 1986; 233: 859-866Crossref PubMed Scopus (748) Google Scholar). Later studies revealed that a large number of proteins include C2 domains, with >200 such proteins in the human genome alone (2International Human Genome Sequence, ConsortiumNature. 2001; 409: 860-921Crossref PubMed Scopus (17336) Google Scholar). We observed that in synaptotagmin 1, a synaptic vesicle protein that binds Ca2+ and contains two C2 domains (3Perin M.S. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (645) Google Scholar), C2 domains are autonomously folded Ca2+-binding modules (4Davletov B.A. Südhof T.C. J. Biol. 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Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Südhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (473) Google Scholar)). Although the Ca2+-binding properties of many C2 domain proteins remain to be examined, the large number of C2 domain proteins in the vertebrate genome makes it likely that this domain represents the second most common Ca2+ binding motif after the EF-hand motif. Most C2 domain proteins are either signal transduction enzymes, such as protein kinase C, or membrane trafficking proteins, such as synaptotagmin 1. At least some isoforms of all major signal transduction enzymes, from ubiquitin ligases to kinases to various phospholipases, contain a C2 domain. Without exception, these proteins are soluble cytosolic enzymes that include a single C2 domain. In contrast, membrane trafficking proteins generally include at least two C2 domains, although a few proteins such as the γ-RIM isoforms (9Wang Y. Südhof T.C. Genomics. 2003; 81: 126-137Crossref PubMed Scopus (82) Google Scholar) and some splice variants of piccolo/aczonin and intersectin (10Wang X. Kibschull M. Laue M.M. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1999; 147: 151-162Crossref PubMed Scopus (152) Google Scholar, 11Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 12Pucharcos C. Casas C. Nadal M. de la Estivill X. Luna S. Biochim. Biophys. Acta. 2001; 1521: 1-11Crossref PubMed Scopus (52) Google Scholar) contain only a single C2 domain. In membrane trafficking proteins the different C2 domains often feature conserved sequence differences, indicating that the C2 domains are functionally specialized. For example, although in synaptotagmin 1 the C2A and C2B domains both bind Ca2+ and phospholipids with similar affinities (13Sugita S. Shin O-H. Han Y. Südhof T.C. J. 2002; PubMed Scopus Google Scholar), the C2B domain contains an that is from the C2A domain but is conserved in all of the C2B domains of J. Shin O-H. Y. Südhof T.C. J. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). trafficking proteins with multiple C2 domains either transmembrane multiple C2 domain and transmembrane region transmembrane multiple C2 domain and transmembrane region or a single either at the (e.g. T.C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) or the C (e.g. Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the few Ca2+ as the major second in with by the of Ca2+ In vertebrate proteins EF-hand Ca2+-binding are common C2 domain proteins (2International Human Genome Sequence, ConsortiumNature. 2001; 409: 860-921Crossref PubMed Scopus (17336) Google Scholar). However, Ca2+ binding in EF-hand proteins often not a function but instead in Ca2+ (e.g. and or a in A. S. Biol. 2000; PubMed Scopus Google and N. H. R. Biol. Chem. 2000; Scholar). the EF-hand proteins with a Ca2+-binding particular to Ca2+-dependent all EF-hand proteins and is in as an sequence from multiple genes in Cell 2000; PubMed Scopus Google and Cell Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). to be different for C2 domain All functionally proteins a C2 domain that binds Ca2+ as Ca2+ and single C2 domain protein the Ca2+-dependent of a to the of in it is to all C2 domain a of the Ca2+-dependent properties of different C2 domain proteins Ca2+ In of these we for conserved C2 domain proteins that function as Ca2+ and C2 domain proteins these are most likely in membrane and data of the for and were for multiple C2 domain proteins, sequence splice variants in sequences, and the genes of MCTPs the with The MCTP were to and and of the human and domains were and by All proteins were as proteins, as Dixon J.E. PubMed Scopus Google Scholar), with and with and high to the that to C2 domains and their properties J. Y. Südhof T.C. J. 2001; PubMed Scopus Google Scholar). and of of MCTP2 to the of not to a splice with to a splice observed in in and were by and were three and of 1 1 and a from to were the and the at for to and The at for 1 and membrane and were All were to the and a phospholipid binding were with soluble proteins in and The C2 domain proteins were with of defined phospholipid in variable of or to defined of or for from For phospholipids from were in and The were by the of C2 domains with with bound C2 domains were by as J. Shin O-H. Y. Südhof T.C. J. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, O-H. J. Südhof T.C. 2002; PubMed Scopus Google Scholar), and bound proteins were in of and analyzed by and domain proteins in 1 of and were with of and in by for at and by by three with of for 1 at and the absence of by C2 domain proteins were for of fluorescence after B. were in an with an of For Ca2+ of Ca2+ were to the from a and fluorescence monitored at the The of Ca2+ with the and the data were analyzed of the in tryptophan fluorescence by the of an of to human MCTPs were the proteins and All proteins were as proteins, but the by the proteins were as for Cell and were in and in with at and after were with the after with were in for and in an for were a a were with of (13Sugita S. Shin O-H. Han Y. Südhof T.C. J. 2002; PubMed Scopus Google Scholar, O-H. J. Südhof T.C. 2002; PubMed Scopus Google Scholar). were with for with and by a of C and and with Cell were by at for and the with of C of in of in C, and of C2 domain protein with various of Ca2+ to the by were for at and and the were at for at and in were analyzed by and were Nature. PubMed Scopus Google Scholar, Jahn R. Südhof T.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were by of MCTP for proteins C2 domains and transmembrane regions, we classes of evolutionarily conserved proteins as synaptotagmins, which are in at least isoforms and are defined by the presence of a single N-terminal and two C-terminal C2 domains with sequence in T.C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google which contain C2 domains and a C-terminal in Cell Biol. Full Text Full Text PDF PubMed Scopus Google and two novel protein that C2 domains with either or C-terminal in We to the proteins an N-terminal and multiple C2 domains as the of these proteins that of of the family as an membrane protein of Biochim. Biophys. Acta. 1999; PubMed Scopus Google these proteins be in a The the second of novel C2 domain proteins, the proteins to as MCTPs multiple C2 domain and at least some splice variants of these proteins contain multiple in to the C2 domains We MCTPs in Caenorhabditis elegans revealed that the C. elegans MCTP is an whose to Y. M. A. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and we a of MCTPs as a their We and for human MCTPs sequence and human revealed that all vertebrates contain two MCTP genes with the and a high of sequence whereas invertebrate elegans and Drosophila contain a single MCTP For comparison of we the human MCTP1 and MCTP2 and the Drosophila and C. elegans MCTP The Drosophila were from two in the data and Although Drosophila MCTP is from the genome sequence to represent two the of the Drosophila sequence with the human and C. elegans and the sequence not that this represents a single The sequence that the of MCTPs are highly both human MCTP1 and C. elegans MCTP are with two to as and and as and the genes contain two that all MCTPs contain three C2 domains to as and in in by two transmembrane regions in short the three C2 domains, whereas a region the C2 domains to the domain are in in The sequence that the two is short and highly All of the MCTP except for the N-terminal region are highly with the of of MCTPs in the domain, the second and the short C-terminal sequence. The a that is in all and isoforms MCTPs transmembrane for MCTPs is the absence of an N-terminal signal in the MCTP and the that all C2 domains are this MCTPs are the only C2 domain proteins that include of the MCTP from sequence of at least two of the that the C2A and C2B domain in we observed in multiple of the a of this some for both MCTP1 and MCTP2 only the second this the conserved C-terminal sequence of MCTPs a sequence, the of the C Although the of MCTPs all the of a high of of is not To whether is of MCTPs a we that proteins of MCTP2 In these proteins, is to the C-terminal of MCTP2 either both or only the or second these proteins, the a of the to the sequence of the sequence alternatively MCTP variants that the We the and whether the presence of either the protein as for a protein we found that but to MCTP2 either of the two it We the of the proteins in All three proteins were to that by is to the protein to and indicating that MCTP2 be a protein of primary of human MCTP1 and MCTP2 and Drosophila MCTP and C. elegans MCTP that are at the in at least two of the are with a as a C2 domain a domain a a the and a C-terminal sequence. are the that the C. elegans sequence a at number that is from the in the the C2A and C2B domains are of the C-terminal of The and two variants for MCTP1 number and and for MCTP2 number are that the splice variants but an of the of the C of primary of human MCTP1 and MCTP2 and Drosophila MCTP and C. elegans MCTP that are at the in at least two of the are with a as a C2 domain a domain a a the and a C-terminal sequence. are the that the C. elegans sequence a at number that is from the in the the C2A and C2B domains are of the C-terminal of The and two variants for MCTP1 number and and for MCTP2 number are that the splice variants but an of the of the C of proteins multiple C2 domains anchored to by a transmembrane classes of proteins were in data to be in the and multiple C2 domain and proteins or are in a membrane by transmembrane regions with and the C2 and all and C are In the of the two major C-terminal splice variants in the sequence in either two are or of the two is C2 domains were the conserved domain data of the some proteins, C2 domains that not the consensus sequence as as transcripts with C2 of proteins in proteins by the in were in and the were with to the were in a to the fluorescence and fluorescence are for at the of the to all of the MCTP and the we the and of the human MCTP MCTP1 is by a large whereas MCTP2 is by a The C-terminal of the which the most sequence contains the in the number and of are observed in the N-terminal of the proteins and The different N-terminal observed in and are by to as and that are by in the genome Most the sequence of the MCTPs at the most often the that The the that we The of the C2A and C2B domains is to the presence or absence of in MCTP1 and and that of the is to the presence or absence of in MCTP1 and in of the human MCTP1 are the of human genome in the and to of the and are and in a of the human MCTP2 are the of human genome in the and to of the and are and in a of revealed that the sequence MCTP1 and were from a large number of such as and To the protein by the MCTP is actually and determine which contain the of these proteins, we two proteins from MCTP1 and MCTP2 of various revealed that MCTP1 is highly in and to a in whereas MCTP2 primarily in and The for MCTP1 are likely to be an of the protein two different to MCTP1 the whereas only of the two MCTP2 MCTP1 in to be in of of the of the the not the of a of of MCTP1 in the that by the of MCTPs are in and that MCTPs are not in with a large number of of the MCTP C2 the three closely spaced C2 domains are the major feature of the of these domains determine the function of these in C. elegans revealed that the MCTP is an and that to Y. M. A. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). but not C2 domains function as Ca2+-binding modules in J. Südhof T.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a the function of we C2 domains. we the two major with C2 domains, Ca2+ binding and with phospholipid of the C2 domain of MCTPs that all three C2 domains to C2 domains. 1 and C2 domains similar structures composed of an but in In C2 domains, the to the of 1 C2 domains is to the of the C2 domain, this the to as a the of C2 domains represents a of the of 1 C2 domains J. Südhof T.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), with the and C the of the domain in 1 C2 domains and at the of the domain in C2 domains. Although all MCTP C2 domains to are not similar The and domains are conserved various MCTP but the of C2 domains is Most C2 domains bind Ca2+ with a intrinsic binding affinity in the absence of phospholipid but with a high apparent affinity in the presence of phospholipid from the that the for bound Ca2+ are in these C2 domains in the absence of phospholipids but are by the phospholipid The Ca2+ binding of C2 domains for the C2 domain from protein kinase C X. Davletov B.A. R.B. Südhof T.C. J. Science. PubMed Scopus Google Scholar, N. S. J. 1999; PubMed Scopus Google Scholar), the C2A and C2B domains of synaptotagmin 1 R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (598) Google Scholar, J. Shin O-H. Y. Südhof T.C. J. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Y. Südhof T.C. J. 2001; PubMed Scopus Google Scholar), and the C2 domain from M. PubMed Scopus Google Scholar). In all of the C2 domains Ca2+ is bound by the from the of the Ca2+ is by or that are in the All of these are conserved in the MCTP C2 domains, the Drosophila and C. elegans MCTP C2 domains, except for the C2B domain of which two of the Ca2+-binding In the conserved of the Ca2+-binding of C2 domains are in the MCTP domains (e.g. the sequence of the that is in all J. Südhof T.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), indicating that these domains the of binding domains. However, H. Shin O-H. M. Südhof T.C. J. Biol. PubMed Scopus Google Scholar) revealed that the Ca2+ binding properties of C2 domains be from sequence the C2B domain of synaptotagmin a perfect Ca2+-binding consensus sequence, intrinsic or the of whether MCTPs function as Ca2+-binding of the MCTP C2 whether the MCTP C2 domains bind to phospholipids as C2 domains. We all three C2 domains of MCTP1 and the domain of MCTP2 as We the binding of these domains to with different phospholipid that neutral and negatively charged phospholipids in the presence of and a that Ca2+-dependent phospholipid binding by the of C2 domain protein that be with in the presence of Ca2+ but not O-H. J. Südhof T.C. 2002; PubMed Scopus Google Scholar). binding is in the or of C2 domains to with the that we (4Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar) and not such as the binding of the synaptotagmin C2B domain J. Shin O-H. Y. Südhof T.C. J. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Surprisingly, the we Ca2+-dependent binding of MCTP C2 domain to of the phospholipid binding for the domain of but this binding the observed with phospholipid-binding C2 domains. that MCTPs are not phospholipid-binding for either charged or neutral the of whether are at all in Ca2+-binding H. Shin O-H. M. Südhof T.C. J. Biol. PubMed Scopus Google Scholar). to MCTP C2 Ca2+ binding to MCTP1 C2 domains, we the C2 domains as proteins, the with and their intrinsic tryptophan fluorescence as a function of Ca2+ by the that all MCTP C2 domains contain a tryptophan in the of and that the C2B domains contain a tryptophan in and in the N-terminal sequence. The tryptophan fluorescence of the C2 domains in the number of fluorescence and their In C2 domain, the of a of Ca2+ the intrinsic tryptophan fluorescence in a that the of The fluorescence large for the C2A and the C2B domains and for the MCTP1 domain. For this we the domain from the domain of this contains an tryptophan and we this domain in all as the MCTP2 domain a Ca2+-dependent fluorescence Ca2+ in and tryptophan fluorescence of the C2 domains not We the tryptophan fluorescence to Ca2+ binding Ca2+-dependent in tryptophan fluorescence were observed for all C2 domains, with and Ca2+. The for the C2A and C2B domains, but the in the domains were However, multiple similar indicating that the observed to a Ca2+ binding of MCTP C2 from properties of the MCTP C2 domains, high affinity intrinsic Ca2+ binding and phospholipid are The apparent Ca2+ affinity of the C2 domains to that of from In studies we that C2 domains with an apparent high Ca2+ affinity in phospholipid from O-H. J. Südhof T.C. 2002; PubMed Scopus Google Scholar). However, for C2 domains, phospholipid binding to a in apparent Ca2+ phospholipid binding and Ca2+ binding are R. A. J. Brose N. J. Rosenmund C. Südhof T.C. Nature. 2001; PubMed Scopus Google Scholar). the MCTP C2 domains the Ca2+ affinity to with but not bind function in Ca2+-dependent To this we the of MCTP C2 domains with of synaptotagmin C2 domains are of in We observed a of the synaptotagmin C2 domains but by the MCTP C2 domains. that with the of MCTPs MCTPs are not of the these that for in the a high Ca2+ binding affinity is not for an In the we describe a novel family of C2 domain proteins that unusual properties of a in Ca2+ which MCTPs from C2 domain proteins, are in the two the of the MCTPs is in that are composed of a sequence primarily composed of three C2 domains and two the MCTPs are now of multiple C2 domain proteins MCTPs are the only C2 domain proteins that contain two and the properties of MCTPs are in that bind Ca2+ but not We only this for MCTP1 and for the domain of but the C2A domain of MCTP2 is highly to that of MCTP1 and thus is likely to bind Ca2+. The C2B domain of the consensus sequence for C2 domain Ca2+-binding and is to bind Ca2+. MCTPs from C. elegans and melanogaster contain all Ca2+-binding with the that Ca2+ binding is evolutionarily conserved and that the C2B domain of MCTP2 is the only C2 domain of an MCTP that not bind Ca2+. these MCTPs are Ca2+-binding in which three Ca2+-binding C2 domains are to the The high affinity of all three MCTP1 C2 domains for Ca2+ phospholipid binding We that the C2B domain of synaptotagmin contains a perfect Ca2+-binding with a high of sequence to that of the C2B domain of synaptotagmin 1, it not bind Ca2+ H. Shin O-H. M. Südhof T.C. J. Biol. PubMed Scopus Google the presence of a Ca2+-binding is not to the Ca2+ binding properties of a C2 domain. In of this the data that the presence of a consensus Ca2+-binding in a C2 domain not the of the of Ca2+ The for C2 domains is to bind Ca2+ in a with phospholipids in which the phospholipid are to the of the bound Ca2+ R. A. J. Brose N. J. Rosenmund C. Südhof T.C. Nature. 2001; PubMed Scopus Google Scholar). The data that this of Ca2+ binding is not in that the MCTP Ca2+-binding for Ca2+ in the absence of The of the MCTP C2 domains, as to this is that in the of the C2 domains, negatively charged in to the the Ca2+ However, negatively charged are that this it is that Ca2+ bind two C2 domains, with the by from the of two different C2 domains. The Ca2+ binding properties and structures of MCTPs that these proteins function in Ca2+ at the the of this function a that the that in C. elegans MCTP is an whose Y. M. A. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of MCTP is with an in Ca2+ that in of a function or an as a Ca2+ Although a Ca2+ function be a Ca2+ function the generally Ca2+ the Ca2+ and the of phospholipid by C2 domains in J. Südhof T.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), C2 domain proteins are EF-hand proteins for some Ca2+ and for Ca2+ In the of this not of their high Ca2+ affinity in the absence of In MCTPs are C2 domain proteins contain two of which be in and in both isoforms that the the of the second is to the C2 domains to the membrane and but the C-terminal sequence of MCTP an sequence is that of the of proteins that contain both C2 domains and a the two that functionally and are in membrane in T.C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of the to as or of their to not the of of their and the family of the MCTPs The that and are in membrane the that MCTPs in membrane the presence of two evolutionarily conserved to the C2 domains to the and the presence of three C2 domains with conserved a Ca2+ to this We A. and E. for