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© The Rockefeller University Press, 0021-9525/1997//847 $5.00
The Journal of Cell Biology, Volume 137, Number 4, , 1997 847-857

Article

Connexin46 Is Retained as Monomers in a trans-Golgi Compartment of Osteoblastic Cells



Michael Koval, James E. Harley, Elizabeth Hick, and Thomas H. Steinberg

Department of Medicine, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

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.

1. Abbreviations used in this paper: BCS, bovine calf serum; BFA, brefeldin A; NRK, normal rat kidney; PVDF, polyvinylidene difluoride.

This research was supported in part by National Institutes of Health grants GM45815 and DK46686.

Please address all correspondence to Michael Koval, Department of Medicine, Washington University School of Medicine, Campus Box 8051, 660 S. Euclid Avenue, St. Louis, MO 63110. Tel.: (314) 362-6606. Fax: (314) 3629230. e-mail: koval{at}id.wustl.edu

As of July 1, 1997, M. Koval's address is Institute for Environmental Medicine and Department of Physiology, University of Pennsylvania School of Medicine, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104.


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