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Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Koval M, Harley JE, Hick E, Steinberg TH - J. Cell Biol. (1997)

Bottom Line: In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers.These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell.Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. koval@id.wustl.edu

ABSTRACT
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

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Expression of Cx46 by transfected Hela cells. (a) Samples corresponding to Hela cells (lane 1) and Hela/Cx46 cells  (lane 2) were normalized for total protein, resolved by SDS/PAGE,  electrophoretically transferred to PVDF, and then immunoblotted for Cx46. Faint nonspecific bands present in both lanes are  also observed in Western blots using preimmune serum (not  shown). (b and c) Untransfected Hela cells (b) or Hela/Cx46 cells  (c) plated on glass coverslips were fixed, permeabilized, and then  immunolabeled with anti-Cx46 and rhodamine-conjugated goat  anti–rabbit IgG. Both images were obtained using the same exposure conditions. Hela/Cx46 cells showed Cx46 present both on  the cell surface (arrows) and in the perinuclear region of the cell.
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Figure 4: Expression of Cx46 by transfected Hela cells. (a) Samples corresponding to Hela cells (lane 1) and Hela/Cx46 cells (lane 2) were normalized for total protein, resolved by SDS/PAGE, electrophoretically transferred to PVDF, and then immunoblotted for Cx46. Faint nonspecific bands present in both lanes are also observed in Western blots using preimmune serum (not shown). (b and c) Untransfected Hela cells (b) or Hela/Cx46 cells (c) plated on glass coverslips were fixed, permeabilized, and then immunolabeled with anti-Cx46 and rhodamine-conjugated goat anti–rabbit IgG. Both images were obtained using the same exposure conditions. Hela/Cx46 cells showed Cx46 present both on the cell surface (arrows) and in the perinuclear region of the cell.

Mentions: Polyclonal serum that recognized Cx46 was initially identified by immunoblot analysis of rat lens total protein preparations that showed reactive bands with the expected Mr at 53 and 68 kD, consistent with previous reports (26, 29, 57). Preincubation of anti-Cx46 antiserum with antigen eliminated binding to both bands in samples prepared from lens and ROS cells (see Fig. 3, lanes 2 and 4). The perinuclear staining pattern obtained for osteoblasts labeled with anti-Cx46 (e.g., see Fig. 2) was eliminated by preincubation of the antiserum with Cx46-his protein (not shown). Also, cells that do not express Cx46 did not show any labeling by immunofluorescence or immunoblotting (see Fig. 4).


Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Koval M, Harley JE, Hick E, Steinberg TH - J. Cell Biol. (1997)

Expression of Cx46 by transfected Hela cells. (a) Samples corresponding to Hela cells (lane 1) and Hela/Cx46 cells  (lane 2) were normalized for total protein, resolved by SDS/PAGE,  electrophoretically transferred to PVDF, and then immunoblotted for Cx46. Faint nonspecific bands present in both lanes are  also observed in Western blots using preimmune serum (not  shown). (b and c) Untransfected Hela cells (b) or Hela/Cx46 cells  (c) plated on glass coverslips were fixed, permeabilized, and then  immunolabeled with anti-Cx46 and rhodamine-conjugated goat  anti–rabbit IgG. Both images were obtained using the same exposure conditions. Hela/Cx46 cells showed Cx46 present both on  the cell surface (arrows) and in the perinuclear region of the cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139843&req=5

Figure 4: Expression of Cx46 by transfected Hela cells. (a) Samples corresponding to Hela cells (lane 1) and Hela/Cx46 cells (lane 2) were normalized for total protein, resolved by SDS/PAGE, electrophoretically transferred to PVDF, and then immunoblotted for Cx46. Faint nonspecific bands present in both lanes are also observed in Western blots using preimmune serum (not shown). (b and c) Untransfected Hela cells (b) or Hela/Cx46 cells (c) plated on glass coverslips were fixed, permeabilized, and then immunolabeled with anti-Cx46 and rhodamine-conjugated goat anti–rabbit IgG. Both images were obtained using the same exposure conditions. Hela/Cx46 cells showed Cx46 present both on the cell surface (arrows) and in the perinuclear region of the cell.
Mentions: Polyclonal serum that recognized Cx46 was initially identified by immunoblot analysis of rat lens total protein preparations that showed reactive bands with the expected Mr at 53 and 68 kD, consistent with previous reports (26, 29, 57). Preincubation of anti-Cx46 antiserum with antigen eliminated binding to both bands in samples prepared from lens and ROS cells (see Fig. 3, lanes 2 and 4). The perinuclear staining pattern obtained for osteoblasts labeled with anti-Cx46 (e.g., see Fig. 2) was eliminated by preincubation of the antiserum with Cx46-his protein (not shown). Also, cells that do not express Cx46 did not show any labeling by immunofluorescence or immunoblotting (see Fig. 4).

Bottom Line: In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers.These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell.Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. koval@id.wustl.edu

ABSTRACT
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

Show MeSH
Related in: MedlinePlus