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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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Cx43 and Cx46 are retained in the same compartment  in the presence of monensin. ROS cells on glass coverslips were  preincubated in MEM containing 10 μM monensin for 4 h at 37°C.  The cells were then fixed, permeabilized, and immunolabeled for  Cx43 (a) and Cx46 (b) as described in Fig. 1.
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Figure 10: Cx43 and Cx46 are retained in the same compartment in the presence of monensin. ROS cells on glass coverslips were preincubated in MEM containing 10 μM monensin for 4 h at 37°C. The cells were then fixed, permeabilized, and immunolabeled for Cx43 (a) and Cx46 (b) as described in Fig. 1.

Mentions: Treatment of NRK cells with BFA inhibits the oligomerization of Cx43, suggesting that Cx43 is assembled into oligomers in a late Golgi apparatus compartment (40). If this is the case, then treatment of ROS cells with monensin, which inhibits cis to medial transport through the Golgi apparatus, should also inhibit Cx43 oligomerization. As shown by immunofluorescence microscopy in Fig. 10, monensin-treated ROS cells showed Cx43 and Cx46 retained in a similar perinuclear region of the cell. This is consistent with results obtained with monensin-treated cardiac myocytes (45). When oligomerization of Cx43 was examined in monensin-treated cells, only 8% (n = 2) of the Triton-soluble Cx43 isolated from monensin-treated cells was assembled into multimers, as compared with control cells that had two- to threefold more Cx43 in the hemichannel form (Fig. 11). Also, since we could detect this difference in the amount of Cx43 assembled into hemichannels, it is likely that nonspecific aggregation of Cx43 did not occur during the solubilization and sucrose gradient fractionation procedure.


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)

Cx43 and Cx46 are retained in the same compartment  in the presence of monensin. ROS cells on glass coverslips were  preincubated in MEM containing 10 μM monensin for 4 h at 37°C.  The cells were then fixed, permeabilized, and immunolabeled for  Cx43 (a) and Cx46 (b) as described in Fig. 1.
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Related In: Results  -  Collection

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

Figure 10: Cx43 and Cx46 are retained in the same compartment in the presence of monensin. ROS cells on glass coverslips were preincubated in MEM containing 10 μM monensin for 4 h at 37°C. The cells were then fixed, permeabilized, and immunolabeled for Cx43 (a) and Cx46 (b) as described in Fig. 1.
Mentions: Treatment of NRK cells with BFA inhibits the oligomerization of Cx43, suggesting that Cx43 is assembled into oligomers in a late Golgi apparatus compartment (40). If this is the case, then treatment of ROS cells with monensin, which inhibits cis to medial transport through the Golgi apparatus, should also inhibit Cx43 oligomerization. As shown by immunofluorescence microscopy in Fig. 10, monensin-treated ROS cells showed Cx43 and Cx46 retained in a similar perinuclear region of the cell. This is consistent with results obtained with monensin-treated cardiac myocytes (45). When oligomerization of Cx43 was examined in monensin-treated cells, only 8% (n = 2) of the Triton-soluble Cx43 isolated from monensin-treated cells was assembled into multimers, as compared with control cells that had two- to threefold more Cx43 in the hemichannel form (Fig. 11). Also, since we could detect this difference in the amount of Cx43 assembled into hemichannels, it is likely that nonspecific aggregation of Cx43 did not occur during the solubilization and sucrose gradient fractionation procedure.

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