Abstract
Protein phosphatase 2A (PP2A) is postulated to be involved in the dephosphorylation of G protein-coupled receptors. In the present study, we demonstrate that the carboxyl terminus of CXCR2 physically interacts with the PP2A core enzyme, a dimer formed by PP2Ac and PR65, but not with the PP2Ac monomer, suggesting direct interaction of the receptor with PR65. The integrity of a sequence motif in the C terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC chemokine receptors, is required for the receptor binding to the PP2A core enzyme. CXCR2 co-immunoprecipitates with the PP2A core enzyme in HEK293 cells and in human neutrophils. Overexpression of dominant negative dynamin 1 (dynamin 1 K44A) in CXCR2-expressing cells blocks the receptor association with the PP2A core enzyme, and an internalization-deficient mutant form of CXCR2 (I323A,L324A) also exhibits impaired association with the PP2A core enzyme, suggesting that the receptor internalization is required for the receptor binding to PP2A. A phosphorylation-deficient mutant of CXCR2 (331T), which has previously been shown to undergo internalization in HEK293 cells, binds to an almost equal amount of the PP2A core enzyme in comparison with the wild-type CXCR2, suggesting that the interaction of the receptor with PP2A is phosphorylation-independent. The dephosphorylation of CXCR2 is reversed by treatment of the cells with okadaic acid. Moreover, pretreatment of the cells with okadaic acid increases basal phosphorylation of CXCR2 and attenuates CXCR2-mediated calcium mobilization and chemotaxis. Taken together, these data indicate that PP2A is involved in the dephosphorylation of CXCR2. We postulate that this interaction results from direct binding of the regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2 after receptor sequestration and internalization.
Highlights
Kines are mediated through interaction with their cognate receptors, which are members of the G protein-coupled receptor (GPCR)1 superfamily
In the process of investigating proteins that interact with chemokine receptors using the yeast two-hybrid system and in vitro binding assay, we discovered that the CXC chemokine receptor, CXCR2, binds to the PR65/PP2Ac dimer but not to the PP2Ac monomer, suggesting direct interaction of the receptor with PR65
We identified the region of CXCR2 involved in the phosphatase 2A (PP2A) binding by using glutathione S-transferase (GST) fusion proteins encoding various fragments of the C terminus of CXCR2 (Fig. 1A)
Summary
Vol 276, No 20, Issue of May 18, pp. 16960 –16968, 2001 Printed in U.S.A. Phosphorylation-independent Association of CXCR2 with the Protein Phosphatase 2A Core Enzyme*. The integrity of a sequence motif in the C terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC chemokine receptors, is required for the receptor binding to the PP2A core enzyme. Pretreatment of the cells with okadaic acid increases basal phosphorylation of CXCR2 and attenuates CXCR2mediated calcium mobilization and chemotaxis Taken together, these data indicate that PP2A is involved in the dephosphorylation of CXCR2. These data indicate that PP2A is involved in the dephosphorylation of CXCR2 We postulate that this interaction results from direct binding of the regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2 after receptor sequestration and internalization. The binding requires the integrity of a sequence motif that is conserved in all CC and CXC chemokine receptors and in many other GPCRs. CXCR2 associates with the PP2A core enzyme in a phosphorylation-independent and internalization-dependent manner
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