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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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Northern blot analysis  of Cx46 mRNA. Total RNA was  isolated from either ROS (lanes  1 and 3) and UMR (lanes 2 and  4) cells, subjected to agarose gel  electrophoresis, transferred to  membranes, and then hybridized  with a radiolabeled cDNA probe  for Cx46 (lanes 1 and 2). The  membranes were then stripped  and reprobed for actin as a control for mRNA loading (lanes 3  and 4). Dashes correspond to  28S and 18S rRNA.
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Figure 1: Northern blot analysis of Cx46 mRNA. Total RNA was isolated from either ROS (lanes 1 and 3) and UMR (lanes 2 and 4) cells, subjected to agarose gel electrophoresis, transferred to membranes, and then hybridized with a radiolabeled cDNA probe for Cx46 (lanes 1 and 2). The membranes were then stripped and reprobed for actin as a control for mRNA loading (lanes 3 and 4). Dashes correspond to 28S and 18S rRNA.

Mentions: By Northern blot analysis, both ROS and UMR cells were found to express Cx46 mRNA (Fig. 1). The intracellular distribution of Cx43 and Cx46 was examined by indirect immunofluorescence (Fig. 2). Consistent with our previous work (30, 53), ROS and primary rat calvarial cells showed high levels of Cx43 expression localized to areas where the cells were in close contact, corresponding to gap junctions. While UMR cells express relatively low levels of Cx43 as compared with ROS cells (53), we were able to locate areas where Cx43 was immunolocalized to the cell surface (Fig. 2 c). In contrast, all three cell types showed expression of Cx46. In each case, Cx46 was predominantly localized to an intracellular compartment in the perinuclear region of the cell.


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)

Northern blot analysis  of Cx46 mRNA. Total RNA was  isolated from either ROS (lanes  1 and 3) and UMR (lanes 2 and  4) cells, subjected to agarose gel  electrophoresis, transferred to  membranes, and then hybridized  with a radiolabeled cDNA probe  for Cx46 (lanes 1 and 2). The  membranes were then stripped  and reprobed for actin as a control for mRNA loading (lanes 3  and 4). Dashes correspond to  28S and 18S rRNA.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Northern blot analysis of Cx46 mRNA. Total RNA was isolated from either ROS (lanes 1 and 3) and UMR (lanes 2 and 4) cells, subjected to agarose gel electrophoresis, transferred to membranes, and then hybridized with a radiolabeled cDNA probe for Cx46 (lanes 1 and 2). The membranes were then stripped and reprobed for actin as a control for mRNA loading (lanes 3 and 4). Dashes correspond to 28S and 18S rRNA.
Mentions: By Northern blot analysis, both ROS and UMR cells were found to express Cx46 mRNA (Fig. 1). The intracellular distribution of Cx43 and Cx46 was examined by indirect immunofluorescence (Fig. 2). Consistent with our previous work (30, 53), ROS and primary rat calvarial cells showed high levels of Cx43 expression localized to areas where the cells were in close contact, corresponding to gap junctions. While UMR cells express relatively low levels of Cx43 as compared with ROS cells (53), we were able to locate areas where Cx43 was immunolocalized to the cell surface (Fig. 2 c). In contrast, all three cell types showed expression of Cx46. In each case, Cx46 was predominantly localized to an intracellular compartment in the perinuclear region of the cell.

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