Phosphoinositide Binding Site Research Articles

Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.

Probing the Phosphoinositide Binding Site of the Clathrin Assembly Protein AP-2 with Photoaffinity Labels

The relative binding specificities of the subunitsof bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tetheredp-benzoyldihydrocinnamoyl-InsPn(BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPnanalogs that sampled the interface between the water and lipid phases, and (iii)sn-1-O-acyl-linked PtdInsPnanalogs that interacted with proteins penetrating the bilayer. The InsPnand PtdInsPnprobes bind with highest selectivity and affinity to the two α subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa μ subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPnbinding site of α-AP-2 prefers more highly phosphorylated species, and (iii) the protein–acyl chain interactions showed high capacity but low selectivity.