Cooperative Regulation of PIP5K by β-Arrestin–GPCR Complexes at Clathrin-Coated Pits Demonstrated by Mathematical Modeling

Background/Objectives: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) is a low-abundance plasma membrane phosphoinositide, an essential factor for clathrin-mediated endocytosis. Activation of Gq-protein-coupled receptors (GqPCR) such as protease-activated receptor 2 (PAR2) triggers phospholipase C–mediated hydrolysis of PIP2, creating a paradox in which the lipid is simultaneously consumed for signaling yet required for receptor internalization. I previously showed that β-arrestin promotes PIP2 regeneration by recruiting Phosphatidylinositol-4-phosphate 5-kinase (PIP5K), partially resolving this paradox. However, the modest hyperactivation of PIP5K (~2–3 fold) and the hump-like kinetics of PIP2 recovery following β-arrestin knockdown indicated that additional regulatory mechanisms were involved. Methods: Here, my revised model incorporates contributions from both preexisting and newly forming clathrin-coated pits (CCPs) using Virtual Cell. Results: I find that CCPs provide a spatially confined environment that promotes clustering of receptor–β-arrestin complexes, thereby amplifying cooperatively PIP5K activity through weak interaction between PIP5K and β-arrestins in the receptor–β-arrestin-CCP complexes. This cooperative mechanism accounts for nonlinear PIP2 recovery kinetics, delayed kinase activation under β-arrestin knockdown conditions, and the sufficiency of modest PIP5K activation to restore plasma membrane lipid pools. These findings suggest that transient and spatially restricted protein interactions, often considered weak or biologically insignificant, play decisive roles in lipid metabolism and receptor trafficking. Conclusions: This framework provides a mechanistic resolution to the paradox of simultaneous PIP2 hydrolysis and receptor internalization and highlights the importance of cooperative dynamics in maintaining plasma membrane signaling competence.