β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂). We found that β-arrestin also helped promote synthesis of PI(4,5)P₂ and up-regulated GPCR endocytosis. We studied these questions with the G_q-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P₂, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P_2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P₂ could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P₂-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P₂ generation that promotes recruitment of PI(4,5)P₂-dependent endocytic machinery.

Membrane phosphatidylinositide lipids (PPIs) are dynamic regulators of diverse cell functions, and their dysregulation underlies numerous human diseases (1). This paper concerns the key involvement of plasma membrane (PM) phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂) in refining receptor–G protein and receptor–β-arrestin coupling (2, 3) and preparing for the endocytosis of receptors (4). Endocytosis requires clustering of adapter proteins on the PM, nucleation of clathrin-coated membrane pits, capture of receptors with β-arrestin (5–7), and pinching off of pits as intracellular vesicles by dynamin GTPase (4, 8–10). In clathrin-mediated endocytosis, PI(4,5)P₂ is typically needed for the assembly of the adaptor protein complexes, clathrin-coated pits (CCPs), and dynamin complexes (4, 11–14). Hence, receptor internalization should be compromised if PI(4,5)P₂ pools are depleted. This raises the question of how receptors that signal by depleting PI(4,5)P₂ can still be internalized. In this study, we found roles of receptor stimulation and β-arrestin in promoting resynthesis of PI(4,5)P₂, thus enabling endocytosis at the PM.Synthesis of PPIs starts with phosphatidylinositol and families of lipid kinases that generate the mono-, bis-, and tris-phosphorylated inositol ring. PM phosphatidylinositol 4-phosphate (PI(4)P) and PI(4,5)P₂ are produced by several mechanisms potentially involving other membrane compartments. They can be synthesized by lipid 4-kinases acting on PM phosphatidylinositol and by lipid 5-kinases acting on PM PI(4)P; they can be delivered in exchange for other lipids by phosphatidylinositol exchange proteins; and they can be delivered through fusion with other membranes (15–23). Such studies show that the PPI pools in different membranes are interdependent (21). For example, depleting PI(4)P locally in the trans-Golgi using a recruitable PI(4)P 4-phosphatase tool reduces the generation of PI(4,5)P₂ at the PM (24). Conversely, depleting PI(4,5)P₂ at the PM by activating muscarinic or angiotensin II receptors also strongly decreases total cellular PI(4)P (25–27). New evidence is emerging that the PPI composition controls membrane trafficking between organelles. For instance, trafficking of mannose 6-phosphate receptors from the Golgi to the PM can be slowed by reduction of PPI synthesis (28) presumably because PPIs are important for fusion of receptor-containing vesicles with the PM.Here, we study contributions of PPI pools to the endocytosis of the G_q-coupled protease-activated receptor 2 (PAR2). This receptor is involved in inflammatory responses (29), sensation of inflammatory pain (30), and cancer metastasis (31). It has been a target of drug development (32) facilitated by recent crystal structures (33). Stimulation of this receptor activates phospholipase C (PLC) to cleave and deplete PI(4,5)P₂ with accompanying production of diacylglycerol, inositol trisphosphate, and calcium signals (34, 35). Activation of the PAR-receptor family has unique properties. The receptor is activated by cleavage of the N terminus by serine proteases such as thrombin, tryptase, or trypsin (34, 36), which generates a tethered N-terminal ligand. The activation stimulates G_q but is followed quickly by desensitization that terminates G_q signaling (34, 35, 37). Our previous experimental results and mathematical modeling suggest that rapid phosphorylation of PAR2 precedes desensitization and that β-arrrestin clamps the phosphorylated and ligand-bound state of the receptor, protecting it from dephosphorylation by serine/threonine phosphatases (38). Then, the receptor is internalized slowly via a clathrin- and dynamin-dependent pathway (8). This rapidly desensitizing receptor is well suited to address mechanisms involved in PPI lipid–dependent G_qPCR endocytosis.Using genetic and optical tools to manipulate and measure PI(4)P and PI(4,5)P₂ levels acutely at the Golgi or the PM, we now demonstrate that PAR2 internalization can be controlled by PM PI(4,5)P₂ that is replenished using both PM and Golgi pools of PI(4)P. A β-arrestin–dependent activation of PIP5-kinase (PIP5K) at the PM turned out to be critical in the formation of PI(4,5)P₂- and PI(4)P-requiring CCPs and potentially other endocytic machinery for receptor internalization.