Abstract
Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C6A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.
Highlights
Growth suppression by connexins can involve intercellular, transmembrane, or intracellular signaling
Together with previously published data, these results suggest that connexin 37 (Cx37) suppresses rat insulinoma (Rin) cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 poreforming domain able to open as a gap junction channels (GJCs)
We have demonstrated that Cx37, but not Cx40 or Cx43, suppresses the proliferation of rat insulinoma (Rin) cells, extending the time spent in each phase of the cell cycle [7]
Summary
Growth suppression by connexins can involve intercellular, transmembrane, or intracellular signaling. Because Cx37 suppresses Rin cell proliferation at low plating densities where cell-cell contact and GJC formation are rare [7, 11] and because Cx37-HCs are active in normal as well as low external [Ca2ϩ] [11], consistent with their activity possibly contributing to growth suppression, we hypothesized that functional Cx37-HCs would be sufficient, and functional GJCs would be unnecessary, for Cx37-mediated suppression of Rin cell proliferation To test this hypothesis we needed a mutation that would prevent docking of otherwise functional HCs. Available data from Cx26 [12, 13] suggested that mutation of any one of the six highly conserved cysteines in the extracellular loops (ECLs) might be sufficient to prevent docking of HCs to form GJCs; data from Cx43 affirmed the importance of ECL cysteines in HC docking and showed that they were not necessary for HC function [14, 15]. These results indicate that 1) the determinants of Cx37 HC function differ from both Cx26 and Cx43, and 2) despite the presence of the full-length C-terminal regulatory domain, Cx37 HC function is not sufficient to successfully suppress Rin cell proliferation
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