title: Rab8 interacts with distinct motifs in alpha2B- and beta2-adrenergic receptors and differentially modulates their transport.
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.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
authors.forename: Chunmin
authors.initials: C
authors.lastname: Dong
authors.forename: Lingling
authors.initials: L
authors.lastname: Yang
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Zhang
authors.forename: Hua
authors.initials: H
authors.lastname: Gu
authors.forename: May L
authors.initials: ML
authors.lastname: Lam
authors.forename: William C
authors.initials: WC
authors.lastname: Claycomb
authors.forename: Houhui
authors.initials: H
authors.lastname: Xia
authors.forename: Guangyu
authors.initials: G
authors.lastname: Wu
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: C547771:ADRA2B protein, human; D018341:Receptors, Adrenergic, alpha-2; D018343:Receptors, Adrenergic, beta-2; D049452:Green Fluorescent Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins