Biomedical subjects

Mitsunobu Ikeda

Publications and source records attributed to Mitsunobu Ikeda.

3 recordsLinked to original sources

PubMed2007

Monoclonal antibody 7F9 recognizes rat protein homologous to human carboxypeptidase-M in developing and adult rat lung.

BACKGROUND AND OBJECTIVE: The purpose of this study was to obtain an antibody that would be useful for investigating the yet unclear molecular mechanism underlying the differentiation of lung alveolar type I and II cells. METHODS: Monoclonal antibodies were raised against membrane proteins from embryonal day 18.5 rat lungs and characterized by immunoblotting on rat lung lysates at various developmental stages to select an appropriate clone. The antigen of the selected antibody was purified by serial column chromatography and immunoprecipitation and identified by mass spectrometry. RESULTS: 7F9 antibody recognizes a 65-kDa protein that is expressed most prominently from embryonal day 20.5 to postnatal day 1. This protein was identified as a rat protein that is similar to 5730456K23Rik protein. The protein is homologous to human carboxypeptidase-M. Although human carboxypeptidase-M is known as a marker of type I cells, the expression of this rat protein was detected in columnar epithelial cells expressing type II cell markers, SP-C and a lamellar body protein ABCA3, in developing lung. Its expression was detected in alveolar cells lacking T1alpha, a type I cell marker protein, in adult lung. It was also expressed in RLE-6TN cells derived from type II cells. The expression in RLE-6TN cells was down-regulated by transforming growth factor-beta1 and up-regulated by Wnt3a. CONCLUSIONS: 7F9 antibody detects a protein in rat lung cells expressing type II markers. The antibody is a useful tool for studying signalling triggered by transforming growth factor-beta1 and Wnt3a in rat type II cells.

Animals↗

PubMed2006

CIN85 is localized at synapses and forms a complex with S-SCAM via dendrin.

Membrane-associated guanylate kinase inverted (MAGI)-1 plays a role as a scaffold at cell junctions in non-neuronal cells, while S-SCAM, its neuronal isoform, is involved in the organization of synapses. A search for MAGI-1-interacting proteins by yeast two-hybrid screening of a kidney cDNA library yielded dendrin. As dendrin was originally reported as a brain-specific postsynaptic protein, we tested the interaction between dendrin and S-SCAM and revealed that dendrin binds to the WW domains of S-SCAM. Dendrin is known to be dendritically translated but its function is largely unknown. To gain insights into the physiological meaning of the interaction, we performed a second yeast two-hybrid screening using dendrin as a bait. We identified CIN85, an endocytic scaffold protein, as a putative dendrin-interactor. Immunocytochemistry and subcellular fractionation analysis supported the synaptic localization of CIN85. The first SH3 domain and the C-terminal region of CIN85 bind to the proline-rich region and the N-terminal region of dendrin, respectively. In vitro experiments suggest that dendrin forms a ternary complex with CIN85 and S-SCAM and that this complex formation facilitates the recruitment of dendrin and S-SCAM to vesicle-like structures where CIN85 is accumulated.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.

Alanine↗