Role of flotillins in the endocytosis of GPCR in salivary gland epithelial cells

Abstract

The muscarinic type 3 receptor (M3R), a GPCR protein located in the plasma membrane, is involved in numerous physiological activities such as smooth muscle contraction and saliva secretion. M3R enters cells through clathrin-mediated endocytosis (CME), while flotillins (flotillin-1 and -2), highly conserved proteins residing in lipid-raft microdomains, make use of clathrin-independent endocytosis (CIE) for their internalization. Since these two proteins use two discrete pathways for endocytic entry, the association of flotillins with CME is poorly understood.

We examined whether flotillins play a role in CME of M3R using immunoblotting, immunocytochemistry, confocal immunofluorescence microscopy, co-immunoprecipitation, and RNA interference techniques in secretory epithelial cells.

Upon stimulation with a cholinergic agonist, M3R, flotillin-1, and flotillin-2 each internalized from the plasma membrane into intracellular sites. The addition of chlorpromazine, filipin III, cytochalasin D, and methyl-β-cyclodextrin (mβCD) affected internalization of M3R and flotillin-1/2 via CME and CIE, respectively. Filipin III and mβCD, lipid raft inhibitors, reduced internalization of M3R slightly, whereas chlorpromazine and cytochalasin D, inhibitors of CME, did not affect endocytosis of the flotillin isoforms. M3R and flotillin-1/2 colocalized and interacted with each other as they entered the cytosol during limited periods of incubation. Interestingly, knockdown of flotillin-1 or -2 by flotillin-specific siRNA prevented internalization and reduced the endocytic efficiency of M3R. Our results suggest that flotillin-1 and -2 are partially involved in CME of M3R by facilitating its internalization.