title: Evolutionarily conserved multiple C2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties.
abstract: 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 Ca(2+)-dependent or Ca(2+)-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 Ca(2+)-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 approximately 1.3-2.3 microM). 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 Ca(2+)-binding but not phospholipid-binding modules.
authors.affiliation: Center for Basic Neuroscience, Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Ok-Ho
authors.initials: OH
authors.lastname: Shin
authors.forename: Weiping
authors.initials: W
authors.lastname: Han
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D005089:Exocytosis; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007438:Introns / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014364:Tryptophan / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D029721:Drosophila Proteins; C496709:MCTP1 protein, human; C496711:MCTP2 protein, human; D008565:Membrane Proteins; D010743:Phospholipids; D014364:Tryptophan; D002118:Calcium