Analysis of the Complex Between the Rem2 GTPase and the L-type Calcium Channel Beta Subunit Suggests a Mechanism for Rem2 Regulation of Insulin Secretion
Abstract Number: 1709-P
Authors: BRIAN FINLIN, DOUGLAS ANDRES, Lexington, KY
Results: Voltage dependant calcium channels (VDCC) play a critical role in regulating insulin secretion from pancreatic beta cells. The Rem2 GTPase has recently been shown to be regulated by glucose in the MIN6 pancreatic beta cell line and to regulate insulin secretion by a mechanism that involves its ability to control the activity of L-type VDCC. The mechanism involves interaction between Rem2 and the cytoplasmic VDCC beta subunit. The beta subunit contains a guanylate kinase (GK) domain that has a high affinity binding site for a cytoplasmic loop of the pore forming alpha subunit called the alpha interaction domain (AID). Beta binding modulates alpha subunit trafficking to the cell surface and also modulates alpha gating properties at the surface. Rem2 could inhibit channel activity by blocking the interaction of beta with AID to prevent alpha trafficking or could bind beta in such a way as to modify alpha properties at the surface. It is therefore important to characterize the Rem2:beta interaction in order to understand the mechanism that Rem2 uses to regulate VDCC. In vitro binding studies reveal that Rem2 binds, in a nucleotide independant manner, to the GK region of the beta subunit which contains the high affinity binding site for AID. However, more refined mapping reveals that Rem2 binds to a region of the GK domain that is distinct from the AID binding domain. Furthermore, competitive inhibition studies reveal that Rem2 does not inhibit the interaction between AID and the beta subunit. Since the Rem2 and AID binding sites on beta are shown to be distinct, it was determined whether Rem2 could bind to the beta subunit while beta was simultaneously bound to AID. These experiments revealed that a Rem2:beta:AID complex could be formed. Taken together these data suggest that Rem2 can form a complex with the channel at the cell surface to potentially modulate its gating properties and does not inhibit beta:AID interaction.