Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein–coupled receptors

Abstract

Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

Structural Development of Cell-Penetrating Peptides Containing Cationic Proline Derivatives

“…Next, we assessed the internalization mechanisms of pep- 15) Therefore, in order to confirm the internalization pathway of peptide 8, the cells were incubated with the tested peptides in the presence of several endocytosis inhibitors (amiloride, 16) an inhibitor of macropinocytosis; nystatin, 17) an inhibitor of caveolae-mediated endocytosis; and sucrose, 18) an inhibitor of clathrin-mediated endocytosis), and the intracellular uptake was measured by flow cytometry (Fig. 5a).…”

Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

“…Clathrin-mediated and caveolae-dependent pathways are the best-characterized internalization routes of GPCRs (Hansen et al ., 1993; Doherty and McMahon, 2009; Guo et al ., 2015). Clathrin-mediated endocytosis (CME) is initiated by the formation of specialized membrane regions called clathrin-coated pits, into which cell-surface receptors concentrate and form clusters.…”

“…In a recent study, clathrin heavy chain- or caveolin1-knockdown cells were employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis (Guo et al ., 2015). The study showed that sucrose, concanavalin A (Con A), and dominant-negative mutants of dynamin blocked other endocytic pathways, as well as the clathrin-mediated pathway.…”

“…In particular, Con A non-specifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, MDC, and dominant-negative epidermal growth factor (EGF)-receptor-pathway substrate 15 (Eps15)-interacting protein (Epsin) mutants are specific for CME when used properly, whereas Pitstop2 is marginally selective for CME (Dutta et al ., 2012; Guo et al ., 2015). These results are summarized in Fig.…”

“…First, the selectivity of inhibitors for specific endocytic routes (e.g., CME or caveolar endocytosis) is always relative (Guo et al ., 2015), and at the same time there could be mutual interactions between endocytic pathways (Rodal et al ., 1999; Subtil et al ., 1999). Second, pharmacological and molecular biological blockade of endocytic processes can inhibit endocytosis of a GPCR, but simultaneously affect other membrane proteins.…”