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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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Connexin hemichannels from Hela/Cx46 cells are stable in Triton X-100. Connexins solubilized from Hela/Cx46 cells  were further incubated at 4°C for 1 h in either the absence (a and  c) or presence (b and d) of 0.2% SDS, and then analyzed by sucrose gradient fractionation as described above. Symbols correspond to measurements of total Cx46 (•) and the 53-kD form of  Cx46 (▪). In the presence of SDS, Cx46 oligomers dissociated  into monomers that migrated on the sucrose gradient as a single  peak centered at 8–9% sucrose.
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Figure 13: Connexin hemichannels from Hela/Cx46 cells are stable in Triton X-100. Connexins solubilized from Hela/Cx46 cells were further incubated at 4°C for 1 h in either the absence (a and c) or presence (b and d) of 0.2% SDS, and then analyzed by sucrose gradient fractionation as described above. Symbols correspond to measurements of total Cx46 (•) and the 53-kD form of Cx46 (▪). In the presence of SDS, Cx46 oligomers dissociated into monomers that migrated on the sucrose gradient as a single peak centered at 8–9% sucrose.

Mentions: One possible concern in using Triton X-100–solubilized preparations is that Cx46-containing oligomers may not be stable under these conditions. To examine this possibility, we prepared Triton X-100–soluble extracts from both Hela/Cx46 cells and whole rat lens. While Hela/Cx46 cells were solubilized using the same conditions used for ROS cells, extensive incubation with 1% Triton X-100 at 4°C was required to solubilize detectable amounts of material from rat lens. Both the 53- and 68-kD forms of Cx46 were solubilized under these conditions (Figs. 12 and 13).


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)

Connexin hemichannels from Hela/Cx46 cells are stable in Triton X-100. Connexins solubilized from Hela/Cx46 cells  were further incubated at 4°C for 1 h in either the absence (a and  c) or presence (b and d) of 0.2% SDS, and then analyzed by sucrose gradient fractionation as described above. Symbols correspond to measurements of total Cx46 (•) and the 53-kD form of  Cx46 (▪). In the presence of SDS, Cx46 oligomers dissociated  into monomers that migrated on the sucrose gradient as a single  peak centered at 8–9% sucrose.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139843&req=5

Figure 13: Connexin hemichannels from Hela/Cx46 cells are stable in Triton X-100. Connexins solubilized from Hela/Cx46 cells were further incubated at 4°C for 1 h in either the absence (a and c) or presence (b and d) of 0.2% SDS, and then analyzed by sucrose gradient fractionation as described above. Symbols correspond to measurements of total Cx46 (•) and the 53-kD form of Cx46 (▪). In the presence of SDS, Cx46 oligomers dissociated into monomers that migrated on the sucrose gradient as a single peak centered at 8–9% sucrose.
Mentions: One possible concern in using Triton X-100–solubilized preparations is that Cx46-containing oligomers may not be stable under these conditions. To examine this possibility, we prepared Triton X-100–soluble extracts from both Hela/Cx46 cells and whole rat lens. While Hela/Cx46 cells were solubilized using the same conditions used for ROS cells, extensive incubation with 1% Triton X-100 at 4°C was required to solubilize detectable amounts of material from rat lens. Both the 53- and 68-kD forms of Cx46 were solubilized under these conditions (Figs. 12 and 13).

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