Phosphorylation-dependent C-terminal Binding of 14-3-3 Proteins Promotes Cell Surface Expression of HIV Co-receptor GPR15

Yukari Okamoto,Sojin Shikano

doi:10.1074/jbc.m110.199695

Abstract

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.

Highlights

Deletion in HIV-1 co-receptor CCR5 (CCR5-⌬32) reduces its cell surface expression and this confers the resistance to viral infection [3]
GPR15 receptor expressed on neutrophils may be involved in neutropenia that correlates with the progress of simian immunodeficiency virus (SIV) [43]
It is well known that the defective cell surface expression of an HIV co-receptor CCR5 results in the long-term nonprogression of HIV infection [44]

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The human GPR15 gene was cloned in pCMV vectors with N-terminal HA or Myc epitope as described previously [19]. For cell surface staining of GPR15 or CD8 in the live cells, the transfected cells were incubated with AF488-Myc mAb at 4 °C for 30 min followed by wash and Hoechst stain. Immunoprecipitation (IP)—For IP of GPR15 from total cell lysate, the transfected cells were washed with PBS once and lysed with lysis buffer (0.5% Igepal, 25 mM Tris, 150 mM NaCl, pH 7.5) containing protease inhibitor cocktails (Pierce) for 20 min at 4 °C. For IP of cell surface GPR15, the cells transfected with Myc-GPR15 constructs were first collected and live stained with Myc mAb for 30 min on ice. After washing, the cells were lysed and the centrifuge supernatants were incubated with protein G-beads for 4 h at 4 °C. The eluted proteins were resolved by SDS-PAGE and blotted for GPR15

RESULTS

C GPR15 Wt

B L360A S359A

DISCUSSION