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(Circulation Research. 2008;102:1539.)
© 2008 American Heart Association, Inc.

Molecular Medicine

The Trafficking Protein GABARAP Binds to and Enhances Plasma Membrane Expression and Function of the Angiotensin II Type 1 Receptor

Julia L. Cook, Richard N. Re, Dawn L. deHaro, Jennifer M. Abadie, Michelle Peters, Jawed Alam

From the Division of Research, Ochsner Clinic Foundation, Ochsner Health System, New Orleans, La.

Correspondence to Dr Julia Cook, Division of Research, Ochsner Clinic Foundation, Ochsner Health System, 1514 Jefferson Hwy, New Orleans, LA 70121. E-mail jcook{at}ochsner.org

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 (AT₁) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT_1A receptor (AT₁R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA_A receptor, as a binding partner for the AT₁R. Interaction of GABARAP with the AT₁R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT₁R 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 AT₁R in PC-12 cells. Cotransfection of GABARAP with an AT₁R 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 AT₁R 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 AT₁R 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 AT₁R to the plasma membrane.

Key Words: angiotensin receptor • AT_1A • GABARAP • yeast 2-hybrid • protein binding