Alternately Translated Product of Cx37 Fails to Arrest Growth in Rin Cells but Alters Cx37 Hemichannel Function when Co‐Expressed

Abstract

Connexin proteins facilitate transmembrane and intercellular communication via channel formation (hemichannel) and intercellular docking of apposed hemichannels to form gap junction channels, respectively. Additionally, they play a role in determining growth phenotype (e.g., arrest, proliferation, death). Connexin 37 (Cx37), expressed in connexin‐deficient rat insulinoma (Rin) cells induces growth arrest or cell death in a manner dependent on the phosphorylation of specific carboxyl terminus (CT) residues. Notably, phosphorylation at serine 319 (S319) is both necessary and sufficient for growth arrest, and mimicking phosphorylation with aspartate substitution at either S275 or S321 is sufficient to induce cell death. 13 of the 21 members of the connexin gene family have a conserved methionine at position 213; in Cx43 mRNA, this methionine codon serves as an alternate translation start site that produces a CT‐spanning translation product. This 20kDa (20k) product interacts with Cx43, chaperoning its trafficking to the membrane. Here we test the hypotheses that 1) this conserved codon in Cx37 mRNA also serves as an alternate translation start site to produce a 13kDa product (13k) that, when expressed alone, regulates growth phenotype of Rin cells and 2) when co‐expressed with Cx37, regulates its channel function. To test these hypotheses, we expressed V5‐tagged 13k or one of two phosphomimetic mutants, 13k‐S321D and 13k‐S275D, in Rin cells or in Rin cells expressing Cx37. Despite a similar phosphorylation profile to Cx37, as determined by mass spectrometry, 13k did not arrest Rin cell growth and neither phosphomimetic mutant induced cell death nor growth arrest. In co‐expressing cells, immunoprecipitation (IP) of 13k failed to co‐IP full‐length Cx37, suggesting no direct interaction between these isoforms; however, 13k and the phosphomimetic mutants altered Cx37 hemichannel conductance and open probability properties, suggesting an indirect interaction of 13k isoforms with Cx37. In conclusion, these data suggest that while 13k and phosphomimetic mutants alone are insufficient to alter the growth phenotype of Rin cells toward growth arrest or death, they nevertheless lead to altered channel properties of full‐length Cx37. The findings herein will inform further research in understanding how connexins are regulated and, in turn, the role they play in regulating cell growth phenotypes.Support or Funding InformationR01 HL131712 | Undergraduate Biology Research Program | University Funds