CONNEXIN46 IS RETAINED AS MONOMERS IN A TRANS-GOLGI COMPARTMENT OF OSTEOBLASTIC CELLS

Connexins are gap junction proteins that form aqueous channels to inte rconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), wh ich 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 jun ction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells w as predominantly localized to an intracellular perinuclear compartment , which appeared to be an aspect of the TGN as determined by immunoflu orescence colocalization. Hela cells transfected with rat Cx46 cDNA (H ela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonph osphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts, To examine connexin assemb ly, 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 onl y the monomer form of Cx46, In contrast, Cx46 expressed by rat lens an d Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related conne xins were differentially regulated in the same cell. Furthermore, olig omerization may be required for connexin transport from the TGN to the cell surface.