Functional expression and biochemical characterization of an epitope-tagged connexin37.
To study the gap junction protein connexin37 (Cx37), we stably transfected cell lines with constructs of human Cx37 containing the epitope tag FLAG (DYKDDDDK). A Cx37 construct containing the FLAG moiety at the carboxyl terminus (Cx37F) was expressed in BWEM cells, and did not substantially alter the levels of endogenous Cx43 in these cells. Immunostaining showed that Cx37F colocalized with Cx43 at cell-cell contacts. Pulse-chase metabolic labeling and immunoprecipitation with anti-FLAG antibodies indicated that Cx37F was synthesized as a protein that ran at 35.9 +/- 0.9 kDa on reducing SDS-PAGE but chased into a slower migrating band at 38.0 +/- 1.0 kDa. This shift in mobility was due to phosphorylation on serine residues, based on [(32)P]-metabolic labeling, immunoprecipitation, and phosphoamino acid analyses. The transition to the phosphoCx37F correlated with a loss of solubility in 1% Triton X-100. Based on the [(35)S]-methionine pulse-chase experiments, the half-life of the labeled Cx37F was approximately 3 h, which is within the range reported for other connexins. Analysis of dye injection experiments indicated that dye transfer was reduced in Cx37-transfected cells in comparison to parental BWEM cells, suggesting that formation of heteromeric Cx37-Cx43 channels reduced the molecular permeability of communication between these cells. Moreover, the similarities of previously demonstrated kinetic details and modification of Cx43 to our new data regarding Cx37 provide evidence for a commonality in processing and assembly steps of these two connexins.
Larson
D M
DM
Mallory Institute of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. davlars@bu.edu
Seul
K H
KH
Berthoud
V M
VM
Lau
A F
AF
Sagar
G D
GD
Beyer
E C
EC
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D017630:Connexins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D051381:Rats; D014162:Transfection; D000097006:Gap Junction alpha-4 Protein
D017630:Connexins; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D000097006:Gap Junction alpha-4 Protein; C085706:FLAG peptide