The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor

Abstract

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 1R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABAA receptor, as a binding partner for the AT 1R. Interaction of GABARAP with the AT 1R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT 1R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT1R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT 1R in PC-12 cells. Cotransfection of GABARAP with an AT 1R fluorescent fusion protein increased PC-12 cell surface expression of the AT1R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT 1R 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 AT1R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT 1R 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 1R to the plasma membrane. © 2008 American heart association, Inc.