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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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Triton solubility of Cx43 and Cx46 in ROS cells. ROS  cells were metabolically radiolabeled with 35S-Trans label, harvested, homogenized, and solubilized in 1% Triton X-100 at either room temperature (lane 1) or 4°C (lanes 2–5) for 30 min. Triton-soluble (lanes 1, 2, and 4) and -insoluble (lanes 3 and 5)  fractions were analyzed by immunoprecipitation using antisera  that recognizes either Cx43 (lanes 1–3) or Cx46 (lanes 4 and 5).  Note that the Cx46 band (lane 4) was somewhat distorted due to  the presence of high levels of IgG heavy chain required for immunoprecipitation. At 4°C, Cx46 showed complete Triton X-100  solubility, while Cx43 was only partially soluble (lane 2). Migration of molecular weight standards is indicated in the figure, and  dots correspond to specific protein bands.
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Figure 8: Triton solubility of Cx43 and Cx46 in ROS cells. ROS cells were metabolically radiolabeled with 35S-Trans label, harvested, homogenized, and solubilized in 1% Triton X-100 at either room temperature (lane 1) or 4°C (lanes 2–5) for 30 min. Triton-soluble (lanes 1, 2, and 4) and -insoluble (lanes 3 and 5) fractions were analyzed by immunoprecipitation using antisera that recognizes either Cx43 (lanes 1–3) or Cx46 (lanes 4 and 5). Note that the Cx46 band (lane 4) was somewhat distorted due to the presence of high levels of IgG heavy chain required for immunoprecipitation. At 4°C, Cx46 showed complete Triton X-100 solubility, while Cx43 was only partially soluble (lane 2). Migration of molecular weight standards is indicated in the figure, and dots correspond to specific protein bands.

Mentions: To examine the assembly of Cx43 and Cx46, we used techniques previously described by Musil and Goodenough (40) using 1% Triton X-100 at 4°C, conditions that enable the solubilization of intact Cx43 hexamers in other cell types. Cx43 and Cx46 were solubilized to different extents (Fig. 8). Note that Cx43 already assembled into gap junctional plaques is resistant to solubilization under these conditions (39). As determined by immunoprecipitation analysis, ∼50% of the Cx43 in ROS cells was soluble in 1% Triton X-100 at 4°C, while virtually all of the Cx46 was solubilized under the same conditions.


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)

Triton solubility of Cx43 and Cx46 in ROS cells. ROS  cells were metabolically radiolabeled with 35S-Trans label, harvested, homogenized, and solubilized in 1% Triton X-100 at either room temperature (lane 1) or 4°C (lanes 2–5) for 30 min. Triton-soluble (lanes 1, 2, and 4) and -insoluble (lanes 3 and 5)  fractions were analyzed by immunoprecipitation using antisera  that recognizes either Cx43 (lanes 1–3) or Cx46 (lanes 4 and 5).  Note that the Cx46 band (lane 4) was somewhat distorted due to  the presence of high levels of IgG heavy chain required for immunoprecipitation. At 4°C, Cx46 showed complete Triton X-100  solubility, while Cx43 was only partially soluble (lane 2). Migration of molecular weight standards is indicated in the figure, and  dots correspond to specific protein bands.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139843&req=5

Figure 8: Triton solubility of Cx43 and Cx46 in ROS cells. ROS cells were metabolically radiolabeled with 35S-Trans label, harvested, homogenized, and solubilized in 1% Triton X-100 at either room temperature (lane 1) or 4°C (lanes 2–5) for 30 min. Triton-soluble (lanes 1, 2, and 4) and -insoluble (lanes 3 and 5) fractions were analyzed by immunoprecipitation using antisera that recognizes either Cx43 (lanes 1–3) or Cx46 (lanes 4 and 5). Note that the Cx46 band (lane 4) was somewhat distorted due to the presence of high levels of IgG heavy chain required for immunoprecipitation. At 4°C, Cx46 showed complete Triton X-100 solubility, while Cx43 was only partially soluble (lane 2). Migration of molecular weight standards is indicated in the figure, and dots correspond to specific protein bands.
Mentions: To examine the assembly of Cx43 and Cx46, we used techniques previously described by Musil and Goodenough (40) using 1% Triton X-100 at 4°C, conditions that enable the solubilization of intact Cx43 hexamers in other cell types. Cx43 and Cx46 were solubilized to different extents (Fig. 8). Note that Cx43 already assembled into gap junctional plaques is resistant to solubilization under these conditions (39). As determined by immunoprecipitation analysis, ∼50% of the Cx43 in ROS cells was soluble in 1% Triton X-100 at 4°C, while virtually all of the Cx46 was solubilized under the same conditions.

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