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Vol. 18, Issue 7, 2473-2480, July 2007

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A Cytosolic Splice Variant of Cab45 Interacts with Munc18b and Impacts on Amylase Secretion by Pancreatic Acini

Patrick P.L. Lam^*, Kati Hyvärinen^,, Maria Kauppi, Laura Cosen-Binker^*, Saara Laitinen, Sirkka Keränen, Herbert Y. Gaisano^*, and Vesa M. Olkkonen

*Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Molecular Medicine, National Public Health Institute, Biomedicum, FI-00251 Helsinki, Finland; Institute of Dentistry, University of Helsinki, FI-00014 Helsinki, Finland; and Technical Research Center of Finland, FI-02044 VTT, Finland

Submitted October 24, 2006; Revised April 3, 2007; Accepted April 5, 2007
Monitoring Editor: Adam Linstedt

We identified in a yeast two-hybrid screen the EF-hand Ca²⁺-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind ⁴⁵Ca²⁺, and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca²⁺. In this assay, Cab45b also binds the Munc18a isoform in a Ca²⁺-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca²⁺-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca²⁺-induced amylase release from streptolysin-O–permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.

Address correspondence to: Herbert Gaisano (herbert.gaisano{at}utoronto.ca) or Vesa Olkkonen (vesa.olkkonen{at}ktl.fi).

Abbreviations used: GST, glutathione S-transferase; SM, Sec1–Munc18 protein; SLO, streptolysin-O; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; syn, syntaxin; ZG, zymogen granule; wt, wild-type.