Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells.
Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore.
S
S
Chen
M C
MC
Liang
J N
JN
Chia
J K
JK
Ngsee
A E
AE
Ting
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015723:Gene Library; D006150:Guanine Nucleotides / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D010911:Pituitary Neoplasms; D017368:Protein Prenylation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D018398:Homeodomain Proteins; D008565:Membrane Proteins; C523657:Pex5l protein, rat; D011994:Recombinant Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; C513177:Rab8b protein, rat; D020691:rab GTP-Binding Proteins