Calmodulin dependence of transferrin receptor recycling in rat reticulocytes

Abstract

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.

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

“…7), indicating that Caz+ influx contributes to Tf-R recycling. This result was in agreement with those in previous papers (Grasso et al, 1990;Morimoto et al, 1995). The similar action of perhexiline, EGTA, BAPTA and Ni2+ on the recycling process may be explained by their interference with Ca".…”

“…Different steps in the endocytosis pathway involve interactions between cytosolic Caz+-binding proteins and membranes. For example, a calmodulin-dependent Tf-R recycling rate was demonstrated in K562 cells (Hunt and Marshall-Carlson, 1986), reticulocytes (Grasso et al, 1990) and MDCK cells (Apodaca et al, 1994). Similarly, calmodulin is involved in the exocytosis process in chromaffin cells (Chamberlain et al, 1995) and in unicellular organisms (Kerboeuf et al, 1993).…”

Hormone-mediated down-regulation of hepatic glutathione synthesis in the rat.

“…Staurosporine (10 nM), a C-kinase inhibitor (25) with Ki of 0.7 nM for C-kinase (per Kamiya Biochem Co.) completely blocked the effect of PMA (500 nM). Calmidazolium, a calmodulin inhibitor with IC50 in the micromolar range (26), was used to examine the importance of the Ca2`-calmodulin pathway. Staurosporine (10 nM) or calmidazolium (1 MM) either alone or together had no effect on GSH synthesis but pretreatment with either agent partially blocked the effect of phenylephrine (0.1 mM).…”

Rapid Activation of Protein Tyrosine Kinase and Phospholipase C-γ2 and Increase in Cytosolic Free Calcium Are Required byEhrlichia chaffeensisfor Internalization and Growth in THP-1 Cells

“…Perhaps one reason is that ehrlichial internalization is dependent on a calcium-dependent enzyme, TGase, and microtubules (40). A second reason is that to accommodate proliferating ehrlichial organisms, new membranes should be added and iron and other nutrients should be supplied by fusion of ehrlichial inclusions with transferrin receptor endosomes (7), which are Ca 2ϩ -calmodulin-and TGase-dependent processes (19,23). A striking finding of the present study was that PLC-␥2 and tyrosine-phosphorylated proteins were rapidly recruited and retained with ehrlichial inclusions even 2 days p.i.…”