RdgB-beta, an interacting partner of the Angiotensin II Receptor-Associated Protein, ATRAP, binds and transfers Phosphatidic Acid

Kathryn Garner1, Alan Hunt2, Grielof Koster2, Pentti Somerharju3, Michelle Li1, Shamshad Cockcroft1. 1University College London, Dept. of Neuroscience, Physiology & Pharmacology, WC1E 6JJ, UK, 2University of Southampton, Clinical & Experimental Sciences, SO16 6YD, UK, 3University of Helsinki, Dept. of Biochemistry & Developmental Biology, Finland

ATRAP, the Angiotensin II Receptor (AT1R)-associated protein, down-regulates angiotensin II signalling by binding to the AT1R, uncoupling it from Gq protein and promoting its internalisation (Daviet et al., 1999; Lopez-Ilasaca et al., 2003). ATRAP interacts with a phosphatidylinositol (PI) transfer protein, RdgBβ (PITPNC1), when cells are stimulated with phorbol myristate acetate (PMA) (Garner et al., 2011), and the present study was undertaken to establish the significance of this interaction for angiotensin II signalling.

RdgBβ binds and transfers phosphatidic acid (PA) in addition to PI. Using a permeabilised [14C]acetate-labelled HL60 cell assay with recombinant transfer proteins and thin layer chromatography (Segui et al., 2002), we observed that RdgBβ binds PI and PA in equal quantities (n = 5), in contrast to another PI transfer protein, PITPα (PITPNA), which binds PI and phosphatidylcholine (PC) equally (n = 6). A dequenching assay (Somerharju et al., 1987) was used to examine PA transfer by RdgBβ: RdgBβ transfers pyrene-labelled PA robustly, across a range of recombinant protein concentrations (5-25μg per assay, n = 3) from quenched donor vesicles (pyrene-PA/ sn1-palmitoyl-sn2-oleoyl-PC (POPC)/ N-trinitrophenyl-phosphatidylethanolamine (TNPE) (2:88:10; mol/mol)) to POPC/POPA (98:2 mol/mol) acceptor vesicles.

Signalling downstream of Gq protein relies on the hydrolysis of PI(4,5)P2 by phospholipase Cβ (PLCβ) to generate the second messengers inositol trisphosphate (IP3), and diacylglycerol (DAG), which can be phosphorylated to PA. In addition, phospholipase D (PLD) produces PA by the hydrolysis of PC. Angiotensin II (100 nM, 30 min) stimulates the incorporation of [3H]inositol into PI ∼3-fold over basal (2.99 ±0.11 s.e.m., n = 3) in HEK-293 cells stably expressing AT1R (HEK-293-AT1R), yet this incorporation was not significantly changed upon over-expression of RdgBβ (2.32 ±0.29 s.e.m, n = 3; t test: p = 0.0969), indicating that RdgBβ does not facilitate flux through the PI cycle when cells are stimulated with angiotensin II.

Mass spectrometric analysis (ESI-MS/MS, Hunt et al., 2004) of the phospholipids bound by RdgBβ indicated that whereas RdgBβ is not selective in its binding of PI, it chooses short-chain saturated or mono-unsaturated PA species (n = 6), consistent with those generated by hydrolysis of PC by PLD, not by PLC. Stimulation of HL60 cells with GTPγS at 10μM Ca2+ for 20mins promoted an increase in PA binding by RdgBβ (1.09 ±0.59 s.d. (pCa7) to 3.34 ±0.18 s.d. (100μM GTPγS, pCa5) relative PA binding per μg protein, n = 2) in contrast to PITPα, whose lipid binding was unaffected by stimulation.

In summary, RdgBβ binds and transfers PLD-derived PA in response to cellular stimulation, and we hypothesise that PA binding stabilises protein expression and provides the signal for binding to ATRAP. Since ATRAP promotes AT1R down-regulation, recruitment of RdgBβ may enable ATRAP to prepare the local lipid environment for receptor endocytosis.

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