The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor.
Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT(1)R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.
Cook
Julia L
JL
Division of Research, Ochsner Clinic Foundation, Ochsner Health System, 1514 Jefferson Hwy, New Orleans, LA 70121, USA. jcook@ochsner.org
Re
Richard N
RN
deHaro
Dawn L
DL
Abadie
Jennifer M
JM
Peters
Michelle
M
Alam
Jawed
J
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D016466:CHO Cells / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000502:physiology*; D019808:Losartan / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification / Q000502:physiology*; D051379:Mice; D008869:Microtubule-Associated Proteins; D016716:PC12 Cells / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D034622:RNA Interference; D011869:Radioligand Assay; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; D003598:Cytoskeletal Proteins; C480861:GABARAP protein, mouse; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; D019808:Losartan