Mechanistic basis of protein phosphatase 2A inhibition by Inhibitor of PP2A (I2PP2A): A key step in βAR resensitization

Abstract: Dysfunction of beta-adrenergic receptor (βAR) is a key hallmark of heart failure, wherein mechanisms of phosphorylation-mediated desensitization of βARs are well understood. However, the dephosphorylation-mediated resensitization of βARs, regulated by protein phosphatase 2A (PP2A), is less understood. We have previously shown that agonist stimulation of βAR activates phosphoinositide 3-kinase γ (PI3Kγ), which phosphorylates the endogenous inhibitor of PP2A (I2PP2A) on serine residues 9 and 93. Phosphorylated I2PP2A (pI2PP2A) inhibits PP2A activity by binding to it and impairs βAR resensitization. Structural studies have shown I2PP2A to be a dimer, yet the functional significance of its dimerization and PP2A inhibition remains unclear. We hypothesize that phosphorylation of I2PP2A promotes its dimerization mediating robust interaction with PP2A leading to PP2A inhibition. To test dimerization, hemagglutinin (HA)- and Myc- tagged I2PP2A were generated and co-expressed in HEK 293 cells. Immunoprecipitation of HA-I2PP2A resulted in significant co-immunoprecipitation of Myc-I2PP2A showing dimerization particularly within the endosomes. Furthermore, fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) experiments showed robust FRET signals, indicating the proximity and colocalization of HA-I2PP2A and Myc-I2PP2A, thereby confirming dimerization. To determine whether phosphorylation is key for dimerization, phospho-mimetic mutants of I2PP2A (S9,93A as inactive and S9,93D as active) were generated and expressed in the transgenic mice. Immunoblotting of tissue lysates expressing these mutants showed that S9,93D primarily formed dimers, whereas S9,93A formed monomers of pI2PP2A suggesting that phosphorylation is a key facilitator of dimerization. Consistent with I2PP2A role in resensitization, I2PP2A knockout cells showed elevated cyclic adenosine monophosphate (cAMP) suggesting restoration of βAR function and resensitization. These findings suggest that I2PP2A dimerization, driven by phosphorylation, impairs βAR resensitization. Therefore, understanding the mechanistic underpinning of the I2PP2A dimerization could offers potential therapeutic strategies for restoring βAR function in heart failure. NIH RO1 grants (HL162772 and HL158746) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.