ABSTRACT
The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+ -dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Calpha functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+ -binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+ -independent C2A domain of double C2 protein gamma (Doc2gamma) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues ((177)RLRRRRR(183)) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma. Because of the presence of the basic cluster, the C2A domain of Doc2gamma did not show Ca2+ -dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.
Subject(s)
Calcium/metabolism , Nuclear Localization Signals/metabolism , Protein Kinase C/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Molecular Sequence Data , Nuclear Localization Signals/chemistry , PC12 Cells , Peptide Mapping , Phospholipids/metabolism , Protein Conformation , Protein Kinase C/chemistry , Rats , Sequence Alignment , Structure-Activity Relationship , Tandem Repeat SequencesABSTRACT
Slp1-3 (synaptotagmin-like protein 1-3) is a new family of carboxyl-terminal-type (C-type) tandem C2 proteins that show higher sequence similarity to the C2 domains of granuphilin-a/Slp-4 than those of other C-type tandem C2 proteins (e.g., synaptotagmin and the Doc2 family). However, the amino (N)-terminal domains of the original Slp1-3 do not contain any known protein motifs and do not show any sequence similarities to each other. We report four alternative splicing isoforms of Slp2 (designated Slp2-a-d, with the original Slp2 renamed Slp2-c) and two alternative splicing isoforms of Slp3 (Slp3-a and Slp3-b, the original Slp3). These isoforms share the same C-terminal tandem C2 structures, but their N-terminal nucleotide sequences are completely different due to the alternate use of different exons. Sequence alignment of the Slp1, Slp2-a, Slp3-a, and Slp4 amino terminal domains reveals the presence of two conserved regions among the Slp family, designated SHD1 (Slp homology domain 1) and SHD2, which may function as protein interaction sites. The SHD1 and SHD2 of Slp3-a and Slp4 are separated by a putative Zn(2+)-binding sequence, whereas Slp1 and Slp2 lack such Zn(2+)-binding sequences and their SHD1 and SHD2 are linked together. In addition, we show that the Slp2-a/c/d mRNAs are differentially distributed in different mouse tissues and at different stages of development, suggesting that these transcripts may be regulated by different promoters.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , DNA Primers/genetics , In Vitro Techniques , Male , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue Distribution , Zinc/metabolismABSTRACT
Na(v)2/NaG is a putative sodium channel, whose physiological role has long been an enigma. We generated Na(v)2 gene-deficient mice by inserting the lacZ gene. Analysis of the targeted mice allowed us to identify Na(v)2-producing cells by examining the lacZ expression. Besides in the lung, heart, dorsal root ganglia, and Schwann cells in the peripheral nervous system, Na(v)2 was expressed in neurons and ependymal cells in restricted areas of the CNS, particularly in the circumventricular organs, which are involved in body-fluid homeostasis. Under water-depleted conditions, c-fos expression was markedly elevated in neurons in the subfornical organ and organum vasculosum laminae terminalis compared with wild-type animals, suggesting a hyperactive state in the Na(v)2-null mice. Moreover, the null mutants showed abnormal intakes of hypertonic saline under both water- and salt-depleted conditions. These findings suggest that the Na(v)2 channel plays an important role in the central sensing of body-fluid sodium level and regulation of salt intake behavior.
Subject(s)
Behavior, Animal/physiology , Central Nervous System/metabolism , Sodium Channels/physiology , Sodium Chloride, Dietary/metabolism , Administration, Oral , Animals , Behavior, Animal/drug effects , Central Nervous System/embryology , Diuretics/pharmacology , Drinking/physiology , Gene Targeting , Genes, Reporter , Heterozygote , Homozygote , Lac Operon , Mice , Mice, Knockout , Molecular Sequence Data , Organ Specificity , Proto-Oncogene Proteins c-fos/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Saline Solution, Hypertonic/administration & dosage , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sodium Channels/genetics , Sulfonamides , Taste/genetics , Thirst/physiology , Voltage-Gated Sodium ChannelsABSTRACT
Gene trapping in embryonic stem (ES) cells was used to identify a novel gene involved in mouse development. In order to screen trapped ES cell lines for the presence of developmentally regulated genes, an in vitro differentiation test was used. One of the G418 resistant cell lines, in conjunction with the lacZ reporter gene, showed differential expression patterns under differentiated and undifferentiated conditions. The gene trap insertion in this cell line was germ-line transmitted and X-gal staining was used to assess the expression pattern of lacZ in embryos heterozygous for the trapped allele. The reporter gene's expression was detected in commissural neurons in the developing spinal cord, suggesting functions for the trapped gene in mouse neural development. Structural analysis of the cDNA revealed that this trapped gene, named PRDC (protein related to DAN and cerberus), is a novel gene that encodes a putative secretory protein consisting of 168 amino acid residues. PRDC gene product shows limited similarities to the products of DAN (differential screening-selected gene aberrative in neuroblastoma) and cerberus. (DAN is a possible tumor-suppressor for neuroblastoma in human. Cerberus can induce an ectopic head in Xenopus embryos when ectopically expressed.) These three gene products may form a novel family of signaling molecules.