Neuronal leucine-rich repeat protein-3 amplifies MAPK activation by epidermal growth factor through a carboxyl-terminal region containing endocytosis motifs.
Neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily. Expression of rat NLRR-3 gene isolated from c-Ha-ras transgenic rat tumor is regulated mainly through the Ras-MAPK signaling pathway. NLRR-3 was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration (0.01 ng/ml) of epidermal growth factor (EGF), but the amplification of MAPK phosphorylation by NLRR-3 was no longer observed when the carboxyl-terminal 30 amino acid stretch containing clathrin-mediated endocytosis motifs was deleted. A green fluorescent protein-tagged NLRR-3 localized at the plasma membrane was efficiently internalized in COS-7 cells, but internalization of a carboxyl-terminal-deleted version (NLRRDeltaC) was less efficient. The presence of clathrin-adaptor protein complexes containing NLRR-3 in brain lysate was confirmed by immunoprecipitation and glutathione S-transferase pull-down experiments, and affinity column chromatography revealed that the carboxyl-terminal region of NLRR-3 interacts with beta-adaptin. We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles.
Fukamachi
Katsumi
K
Experimental Pathology, Chemotherapy Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Matsuoka
Yoichiro
Y
Ohno
Hiroshi
H
Hamaguchi
Tetsuya
T
Tsuda
Hiroyuki
H
D016475:3T3 Cells; D033966:Adaptor Protein Complex beta Subunits / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D004705:Endocytosis; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007930:Leucine / Q000737:chemistry*; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
D033966:Adaptor Protein Complex beta Subunits; C102717:Lrrn3 protein, mouse; C484458:Lrrn3 protein, rat; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D007930:Leucine