GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members.
The tubby gene family consists of four members, TUB, TULP1, TULP2 and TULP3, with unknown function. However, a splice junction mutation within the mouse tub gene leads to retinal and cochlear degeneration, as well as maturity onset obesity and insulin resistance. Mutations within human TULP1 have also been shown to co-segregate in several cases of autosomal recessive retinitis pigmentosa (RP) and TULP1 deficiency in mice leads to retinal degeneration. The primary amino acid sequences of the tubby family members do not predict a likely biochemical function. As a first step in defining their function, we present a detailed characterization of the cellular and subcellular localization of the human (TUB) and mouse (tub) homologous gene products. We report the isolation of TUB splice variants which have different subcellular localizations (nuclear versus cytoplasmic) and which define a nuclear localization signal. In addition, using green fluorescent protein (GFP) tags, we observe a nuclear localization for TULP1, similar to TUB splicing forms TUB 561 and TUB 506. Finally, we report tubby expression in mouse brain by in situ hybridization and by immunohistochemistry with polyclonal antibodies. Protein was found in both the hypothalamic satiety centers and in a variety of other CNS structures including the cortex, cerebellum, olfactory bulb and hippocampus. Both nuclear and cytoplasmic signals were detected with a series of independently generated polyclonal antibodies, consistent with the presence of multiple alternatively spliced isoforms within the CNS.
He
W
W
AXYS Pharmaceuticals, La Jolla, CA 92037, USA.
Ikeda
S
S
Bronson
R T
RT
Yan
G
G
Nishina
P M
PM
North
M A
MA
Naggert
J K
JK
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D017173:Caenorhabditis elegans; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D004285:Dogs; D004330:Drosophila; D005091:Exons; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D005810:Multigene Family; D009765:Obesity / Q000235:genetics; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D005136:Eye Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C105443:TULP1 protein, human; C105444:TULP2 protein, human; C116465:TULP3 protein, human; C098988:Tub protein, mouse; C491353:Tulp1 protein, mouse; C484551:Tulp2 protein, mouse; C485499:Tulp3 protein, mouse; D049452:Green Fluorescent Proteins