Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

Chunmin Dong,Lingling Yang,Xiaoping Zhang,Hua Gu,May L Lam,William C Claycomb,Houhui Xia,Guangyu Wu

doi:10.1074/jbc.m109.081521

Chunmin Dong, Lingling Yang + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.081521

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Abstract

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of alpha(2B)- and beta(2)-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of alpha(2B)-AR than beta(2)-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by alpha(2B)-AR, but not beta(2)-AR, and arrested alpha(2B)-AR in the TGN compartment. Co-immunoprecipitation revealed that both alpha(2B)-AR and beta(2)-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked beta(2)-AR interaction with Rab8, whereas mutation of residues Val(431)-Phe(432)-Asn(433)-Gln(434), Pro(447)-Trp(448), Gln(450)-Thr(451), and Trp(453) in the C terminus impaired alpha(2B)-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric beta(2)-AR carrying the alpha(2B)-AR C terminus was similar to alpha(2B)-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of alpha(2B)-AR and beta(2)-AR and differentially modulates their traffic from the TGN to the cell surface.

Highlights

Rab8 expression was measured by immunoblotting using anti-FLAG and anti-Rab8 antibodies (Fig. 1A) and the cell surface expression of ␣2B-adrenergic receptors (AR) and ␤2-AR was quantified by ligand binding in intact live cells using the membrane-impermeable ligands [3H]RX821002 and [3H]CGP12177, respectively
The molecular mechanism underlying the transport of nascent G protein-coupled receptors (GPCRs) from the Golgi to the functional destination, the plasma membrane, remains poorly understood
Rab8 GTPase, which has been demonstrated to modulate vesicular protein transport from the trans-Golgi network (TGN) to the apical/basolateral plasma membrane, in the cell surface targeting of ␣2B-AR and ␤2-AR

Summary

EXPERIMENTAL PROCEDURES

Materials—Antibodies against phospho-ERK1/2, green fluorescent protein (GFP), and ␣2B-AR were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For measurement of ERK1/2 activation by endogenous ␣2B-AR in NG108 cells, the cells were cultured in 6-well plates and transfected with 2 ␮g of Rab8(T22N) with FuGENE 6 reagent in serum-free medium for. Immunofluorescence Microscopy—For fluorescence microscopic analysis of receptor subcellular localization, HEK293 cells were grown on coverslips pre-coated with poly-L-lysine in 6-well plates and transfected with 50 ng of GFP-tagged receptors with or without co-transfection together with 400 ng of Rab8(T22N). After washing with PBS (3 ϫ 5 min), the cells were incubated with Alexa Fluor 594-labeled secondary antibody (1:2000 dilution) for 1 h at room temperature. Each sample was further diluted 5 times with 1ϫ SDS gel loading buffer, separated by SDS-PAGE, and probed with anti-HA antibodies to determine the amount of the receptor in the immunoprecipitates [19].

RESULTS

We then determined the effect of transient expression of

DISCUSSION