Critical role of the N-terminal cyclic AMP-binding domain of Epac2 in its subcellular localization and function.
cAMP is a well-known regulator of exocytosis, and cAMP-GEFII (Epac2) is involved in the potentiation of cAMP-dependent, PKA-independent regulated exocytosis in secretory cells. However, the mechanisms of its action are not fully understood. In the course of our study of Epac2 knockout mice, we identified a novel splicing variant of Epac2, which we designate Epac2B, while renaming the previously identified Epac2 Epac2A. Epac2B, which lacks the first cAMP-binding domain A in the N-terminus but has the second cAMP-binding domain B of Epac2A, possesses GEF activity towards Rap1, as was found for Epac2A. Immunocytochemical analysis revealed that exogenously introduced Epac2A into insulin-secreting MIN6 cells was localized near the plasma membrane, while Epac2B was found primarily in the cytoplasm. Interestingly, cAMP-binding domain A alone introduced into MIN6 cells was also localized near the plasma membrane. In MIN6 cells, Epac2A was involved in triggering hormone secretion by stimulation with 5.6 mM glucose plus 1 mM 8-Bromo-cAMP, but Epac2B was not. The addition of a membrane-targeting signal to the N-terminus of Epac2B was able to mimic the effect of Epac2A on hormone secretion. Thus, the present study indicates that the N-terminal cAMP-binding domain A of Epac2A plays a critical role in determining its subcellular localization and potentiating insulin secretion by cAMP. J. Cell. Physiol. 219: 652-658, 2009. (c) 2009 Wiley-Liss, Inc.
Niimura
Manabu
M
Division of Cellular and Molecular Medicine, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.
Miki
Takashi
T
Shibasaki
Tadao
T
Fujimoto
Wakako
W
Iwanaga
Toshihiko
T
Seino
Susumu
S
D000311:Adrenal Glands / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002540:Cerebral Cortex / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; C486707:Rapgef4 protein, mouse; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins