Regulation of Folate Receptor Internalization by Protein Kinase C α

Abstract

The glycosyl-phosphatidylinositol anchored folate receptor (FR) mediates selective delivery of a broad range of experimental drugs to the receptor-rich tumors, but molecular mechanisms controlling FR internalization have not been adequately studied. FR quantitatively recycles between the cell surface and endocytic compartments via a Cdc42-dependent pinocytic pathway. Protein kinase C (PKC) activators including diacylglycerol and phorbol ester have previously been reported to increase the proportion of FR on the cell surface. Here we identify the alpha-subtype of PKC as the mediator of phorbol ester action on FR recycling and provide evidence that activated PKCalpha is recruited to FR-rich membrane microdomains where, in association with its receptor RACK1, it inhibits FR internalization; the activation state of Cdc42 remains unaltered. We also show that the PKC substrate, annexin II, is required for FR internalization. The studies clarify a molecular mechanism for the regulation of FR recycling through PKC which could potentially be exploited for effective drug delivery.

Dual Targeting of Epithelial Ovarian Cancer Via Folate Receptor α and the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates

“…For these experiments, cells were washed with acid-buffered (pH 3.5) saline, neutralized, then incubated with 50 nM [ 3 H]folic acid or [ 3 H] AGF154 at neutral pH (pH 7.4) (i) at 4°C to measure surface FRα-bound [ 3 H]substrate. Cells were also incubated at 37°C for 1 h, after which (ii) total cell-associated [ 3 H]substrate (includes sum of both [ 3 H]folic acid or [ 3 H] AGF154 bound to surface FRα and internalized [ 3 H]folic acid or [ 3 H] AGF154 ) and (iii) internalized [ 3 H]substrate were quantified (20, 34). For all treatments, parallel incubations were performed with 10 µM non-radioactive folic acid which competes with FR-mediated binding and uptake so as to identify the non-specific (non-FRα-mediated) radiolabeled fraction.…”

“…For assays of FRα-mediated [ 3 H]folic acid and [ 3 H] AGF154 uptake by IGROV1 NTC and KD-4 and KD-10 FRα knockdown cells (34), cells were seeded at ∼0.8 × 10 6 cells/60 mm dish three days prior to experiment. For uptake assays, the cells were washed at room-temperature 3x with PBS, then washed 2x with acetate buffer (pH 3.5).…”

Folate receptor alpha (FRA) expression in breast cancer: identification of a new molecular subtype and association with triple negative disease

“…In the absence of membrane staining, intracellular staining was rarely present. Intracellular staining for FRA is expected given that the receptor cycles, carrying folates with it, to the intracellular compartment while remaining membrane associated, by an endocytotic mechanism ([Elnakat et al 2009]).…”

Novel functions of folate receptor alpha in CNS development and diseases

“…Elnakat et al, [24] showed that Protein Kinase Cα (PKCα) substrate, annexin II, is required for FR internalization. When activated PKCα is recruited to FR-rich membrane caveolar microdomains, it inhibits FRα internalization.…”

“…Endocytosis of FRα is assisted by low-density lipoprotein (LDL) receptorrelated protein 2 (LRP2), a multifunctional cell-surface receptor expressed in the embryonic neuroepithelium [23] as well as by protein kinase Cα [24].…”