Caveolin-1 Mediates the Internalization of Functional Insulin Receptors in Beta-Cells

Abstract

Type 2 diabetes is associated with defects in insulin receptor (InsR) signalling, including in beta-cells. Internalization of the activated InsR complex is necessary to initiate signalling. Mechanisms of InsR internalization in beta-cells remain unclear, although Caveolin-1 (CAV1) has been implicated in other cell types. We labelled InsR with monomeric fluorescent proteins in a non-critical linker region between the furin-like region and the transmembrane domain. Total internal reflection microscopy in living MIN6 beta-cells revealed that CAV1 is recruited to InsR membrane domains right before receptor internalization. These results were confirmed by super-resolution stimulated emission depletion microscopy, which showed a CAV1 protein coat around membrane nanodomains containing InsR. To investigate if CAV1 is essential for InsR internalization, we compared the distribution of InsR in cells expressing CAV1 mutant proteins. MIN6 cells over-expressing dominant negative CAV1-Y14F showed defects of InsR internalization, with virtually complete accumulation plasma membrane of InsR. Overexpression of CAV1 in MIN6 cells leads to an increased Erk1/2 phosphorylation upon insulin treatment compared to empty-vector controls. Erk1/2 signalling is further increased in cells overexpressing the dominant-positive CAV1-Y14D mutant. Interestingly, Akt phosphorylation was not affected by CAV1 overexpression. These data strongly suggest that CAV1-mediated internalization of the InsR is required for Erk1/2 signalling but Akt signalling is mostly induced by insulin receptors localized at the plasma membrane. Together, our data give new insights on the mechanisms in InsR signalling in the beta-cell.