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A Low-Affinity Ca²⁺-Dependent Association of Calmodulin With the Rab3A Effector Domain Inversely Correlates With Insulin Exocytosis

¹ Pacific Northwest Research Institute and Department of Pharmacology, University of Washington, Seattle, Washington
² Central Research Division, Pfizer, Groton, Connecticut

The stimulus-response coupling pathway for glucose-regulated insulin secretion has implicated a rise in cytosolic [Ca²⁺]_i as a key factor to induce insulin exocytosis. However, it is unclear how elevated [Ca²⁺]_i communicates with the pancreatic ß-cell’s exocytotic apparatus. As Rab3A is a model protein involved in regulated exocytosis, we have focused on its role in regulating insulin exocytosis. By using a photoactivatable cross-linking synthetic peptide that mimics the effector domain of Rab3A and microsequence analysis, we found calmodulin to be a major Rab3A target effector protein in pancreatic ß-cells. Coimmunoprecipitation analysis from pancreatic islets confirmed a Rab3A-calmodulin interaction in vivo, and that it inversely correlated with insulin exocytosis. Calmodulin affected neither GTPase nor guanine nucleotide exchange activity of Rab3A. The calmodulin-Rab3A interaction was pH- and Ca²⁺-dependent, and it was preferential for GTP-bound Rab3A. However, Rab3A affinity for calmodulin was relatively low (K_d = 18–22 µmol/l at 10^-5 mol/l [Ca²⁺]) and competed by other calmodulin-binding proteins that had higher affinity (e.g., Ca²⁺/calmodulin-dependent protein kinase-2 [CaMK-2] {K_d = 300–400 nmol/l at 10^-5 mol/l [Ca²⁺]}). Moreover, the Ca²⁺ dependence of the calmodulin-Rab3A interaction (K_0.5 = 15–18 µmol/l [Ca²⁺], maximal at 100 µmol/l [Ca²⁺]) was significantly lower compared with that of the calmodulin–CaMK-2 association (K_0.5 = 40 µmol/l [Ca²⁺], maximal at 1 mmol/l [Ca²⁺]). The data suggested that a transient Rab3A-calmodulin interaction might represent a means of directing calmodulin to the cytoplasmic face of a ß-granule, where it can be subsequently transferred for activation of other ß-granule–associated calmodulin-binding proteins as local [Ca²⁺]_i rises to promote insulin exocytosis.

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