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

Abstract

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 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. receptors are 7-transmembrane G protein-coupled receptors (GPCRs) of the largest GPCR subfamily, family 1, or the rhodopsin-like family. The GPCR superfamily has more than 860 members 1 and more than 50 "GPCR-associated" proteins have now been discovered, the majority of which interact with GPCR cytoplasmic carboxyl termini. 2 Most of these are involved in trafficking, subcellular targeting, and intracellular signaling. Our preliminary studies were designed to identify proteins that bind to the cytoplasmic carboxyl terminus of the AT 1 receptor (AT 1 R), the most prevalent and best characterized of the Ang receptors. Such proteins are expected to be involved in trafficking of the AT 1 R through the secretory pathway and to the plasma membrane, as well as in ligand-mediated internalization and recycling. Moreover, our recent published studies suggest that the AT 1 R is cleaved in a ligand-dependent manner to liberate the cytoplasmic domain, a significant quantity of which traffics to the nucleus. 3 Presumably, this nuclear trafficking event also involves sequence-specific binding proteins. Using a yeast 2-hybrid (Y2H) approach to screen a mouse brain library, we have ide...

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…Kidney-specific ATRAP1 transgenic mice exhibit hypertension and renal hypertrophy and failure (Oppermann et al, 2010), suggesting that renal ATRAP1 plays an important role in regulating intrarenal RAS. Mechanistic aspects of AT 1 receptor interaction with ATRAP1 and transfer of signal are not clear at this time (Cook et al, 2008).…”

Effects of C-terminal Modifications of GEC1 Protein and γ-Aminobutyric Acid Type A (GABAA) Receptor-associated Protein (GABARAP), Two Microtubule-associated Proteins, on κ Opioid Receptor Expression

“…Because of the high degree of identity, GABARAP and GEC1 are likely to have overlapping functions. The findings that GABA A receptor, KOPR, and AT1 angiotensin II receptor interact with both GEC1 and GABARAP (6,13,20,35) support this notion. In GABARAP-null mice, there is no change in GABA A receptor levels or membrane clustering (43), most likely due to the redundant functions between the two proteins.…”

“…A, one of the four immunoblotting (IB) results obtained. An aliquot of HA-GEC1 or HA-GABARAP from each concentration (10,20,40, and 80 g/ml) was loaded as loading control (upper right) for every experiment and was used to generate standard curves as shown in B. DLU, digital line unit. C, the bound HA-GEC1 or HA-GABARAP was quantified using the standard curves in each data set and plotted against the incubation concentration.…”

“…GEC1 has been shown to interact with GABA A receptor (35), KOPR (7), and AT1 angiotensin II receptor (20) and enhance cell surface expression of the KOPR (7). GABARAP is associated with and increases cell surface expression of GABA A receptor (13,18), KOPR (6), Na ϩ -dependent P i cotransporter, sodium P i IIa (41), AT1 angiotensin II receptor (20), and TRPV1 (21). In addition, GABARAP also binds transferrin receptor (42).…”

“…GATE16 is involved in intra-Golgi transport (14). GABARAP interacts with GABA A and AT1 angiotensin II receptors and transient receptor potential vanilloid 1 (TRPV1) and promotes their cell surface expression (13,18,20,21). Atg8 and LC3 are essential for autophagy, and GABARAP and GATE16 are also shown to be involved.…”