A. Butterweck et al., IMMUNOCHEMICAL CHARACTERIZATION OF THE GAP JUNCTION PROTEIN CONNEXIN45 IN MOUSE KIDNEY AND TRANSFECTED HUMAN HELA-CELLS, The Journal of membrane biology, 141(3), 1994, pp. 247-256
Antibodies to the gap junction protein con nexin45 (Cx45) were obtaine
d by immunizing rabbits with fusion protein consisting of glutathione
S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As s
hown by immunoblotting and immunofluorescence, the affinity-purified a
ntibodies recognized Cx45 protein in transfected human HeLa cells as w
ell as in the kidney-derived human and hamster cell lines 293 and BHK2
1, respectively. In Cx45-transfected HeLa cells, this protein is phosp
horylated as demonstrated by immunoprecipitation after metabolic label
ing. The phosphate label could be removed by treatment with alkaline p
hosphatase. A weak phosphorylation of Cx45 protein was also detected i
n the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenos
ine- or guanosine monophosphate (cAMP, cGMP) did not alter the level o
f Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK2
1 cells. The addition of the tumor-promoting agent phorbol 12-myristat
e 13-acetate (TPA) led to an increased P-32 phosphate incorporation in
to the Cx45 protein in transfected cells. The Cx45 protein was found i
n homogenates of embryonic brain, kidney, and skin, as well as of adul
t lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli
and distal tubules, whereas connexin32 and -26 were coexpressed in pr
oximal tubules. No connexin43 protein was detected in renal tubules an
d glomeruli at this stage of development. Our results suggest that cel
ls in proximal and distal tubules are interconnected by gap junction c
hannels made of different connexin proteins. The Cx45 antibodies chara
cterized in this paper should be useful for investigations of Cx45 in
renal gap junctional communication.