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

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 α 2B ‐ and β 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 α 2B ‐AR than β 2 ‐AR. The GDP‐bound Rab8(T22N) mutant attenuated ERK1/2 activation by α 2B ‐AR, but not β 2 ‐AR, and arrested α 2B ‐AR in the TGN compartment. Co‐immunoprecipitation revealed that both α 2B ‐AR and β 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 β 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 α 2B ‐AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric β 2 ‐AR carrying the α 2B ‐AR C terminus was similar to α 2B ‐AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of α 2B ‐AR and β 2 ‐AR and differentially modulates their traffic from the TGN to the cell surface.