Transferrin Receptor Functions as a Signal‐Transduction Molecule for its Own Recycling Via Increases in the Internal Ca2 Concentration

Transferrin binding to its receptor modulates transferrin receptor (Tf‐R) recycling rates in several cells [Klausner, R. D., Van Renswoude, J., Ashwell, G., Kempf, C., Schechter, A., Dean, A. & Bridges, K. R. (1983a) J. Biol. Chem. 258, 4715–4724; Gironès, N. & Davis, R. J. (1989) Biochem. J. 264, 35–46; Sainte‐Marie, J., Vidal, M., Bette‐Bobillo, P., Philippot, J. R. & Bienvenüe, A. (1991) Eur. J. Biochem. 201, 295–302]. To delineate the mechanism of this regulation, we hypothesized that the binding of the ligand to its receptor could lead to activation of several second‐messenger pathways, which may redundantly stimulate recycling of the receptor. The effects of different regulators of Ca2 flux or concentrations were investigated on the Tf‐R–recycling pathway; these studies were carried out in two cell types. Perhexiline, a calcium antagonist, slowed receptor recycling in comparison with the control by more than 80% in L2C cells and by 60% in Jurkat cells (B and T lymphoblasts, respectively) but did not affect their internalization rate. Perhexiline thus trapped considerable amounts of Tf‐R in the internal compartment. Ca2 chelators, such as EGTA or 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N,N′‐tetraacetic acid, and a Ca2‐channel inhibitor (Ni2) decreased drastically the recycling rate of Tf‐R. Tf‐R recycling was shown to be slowed by a calmodulin antagonist. Conversely, artificial elevation of free internal Ca2 in L2C cells, using lectin, accelerated the recycling rate. These results suggest that the intracellular Ca2 concentration plays an important role in the outward flow of transferrin receptors. Consequently, we examined the role of transferrin in internal free Ca2 regulation. The addition of transferrin or anti‐(Tf‐R) Ig specifically elicited a rise in [Ca2], as demonstrated by inefficacy of apotransferrin or irrelevant antibodies. These results suggest that Ca2 is a regulator of Tf‐R recycling and that Tf‐R seems to function as a signal‐transduction molecule (perhaps in conjunction with other membrane proteins) rather than merely as an endocytic receptor.