Published online before print May 22, 2008, doi: 10.1161/CIRCRESAHA.108.176594
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© 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
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
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Abstract |
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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 AT1A receptor (AT1R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABAA 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 • AT1A • GABARAP • yeast 2-hybrid • protein binding
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Introduction |
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The angiotensin (Ang) II type 1 (AT1) and 2 (AT2) 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 members1 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 AT1 receptor (AT1R), the most prevalent and best characterized of the Ang receptors. Such proteins are expected to be involved in trafficking of the AT1R 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 AT1R 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 identified several proteins which bind to the AT1AR, the most prevalent of which are GABARAP (
-aminobutyric acid [GABA] receptor–associated protein) and the related protein GABARAPL1 (L1 indicates like-1). Of 40 clones isolated, approximately one-half were identified by sequence analysis, as GABARAP or GABARAPL1, both members of the microtubule-associated protein (MAP) family. GABARAP was originally identified through its binding to one subunit of the pentameric ionotropic GABAA receptor. It is involved in trafficking of the GABAA receptor to the plasma membrane via microtubule tracks and affects both clustering and kinetic properties of the receptor. GABA is the major inhibitory neurotransmitter in the brain and acts through the ionotropic GABAA and GABAC receptors and the metabotropic GABAB receptor.4 Of these, GABARAP is known to bind only to the GABAA receptor. Postsynaptic binding of GABA to the GABAA receptor opens chloride ion channels and leads to hyperpolarization, thereby slowing neuroelectrical impulses. Coexpression of GABARAP has been shown to increase the level of GABAA receptors detected at the plasma membrane and to cluster recombinant GABAA receptors,5–8 the net effect of which is to modulate neuroelectrical inhibition.
GABARAPL1 (GEC1), originally identified as an estrogen-induced protein homologous to GABARAP,9 has since been found to bind to the GABAA receptor10 and to the carboxyl terminus (C terminus) of the metabotropic 
-opioid receptor (KOR) and to facilitate receptor trafficking of the KOR from the endoplasmic reticulum/Golgi to the plasma membrane.11 When expressed in CHO cells, GABARAPL1 coimmunoprecipitates with KOR and greatly increases total and cell surface KOR opioid receptors but not µ- or 
-opioid receptors. Both of the MAPs, GABARAP and GABARAPL1, therefore, are involved in plasma membrane-directed protein trafficking.
The vital importance of accessory proteins, such as GABARAP, that are involved in intracellular trafficking is exemplified by the development of kidney hypertrophy and hypertension in transgenic mice overexpressing Ang II receptor–associated protein 1 (ARAP1), a protein that is involved in AT1R "recycling" in the kidney.12 The studies described herein were designed to confirm the observed AT1R:GABARAP interaction in yeast and to investigate the nature and function of the intermolecular association.
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Materials and Methods |
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Y2H Analysis
An adult mouse brain cDNA library cloned into a GAL4 activation domain vector (pGADT7-Rec) and transformed into yeast strain Y187 was obtained from Clontech (catalog no. 638863) and used for Y2H screening as recommended by the manufacturer.
The AT1R C terminus was ligated in-frame into pGBKT7 to produce a Gal4-DBD:AT1R C terminus fusion protein. The Y2H target library consisted of mouse brain sequences ligated into pGADT7-Rec to produce Gal4-AD fusion sequences (library from Clontech) (DBD indicates DNA-binding domain; AD, activation domain).
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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Note that the term GABARAP(X) refers to the combined terms GABARAP and GABARAPL1.
Y2H Screen
A Y2H screen of a mouse brain cDNA library was conducted to identify proteins that interact with the cytoplasmic C terminus of the AT1AR (amino acids 306 to 359). Of 1.3x106 clones screened, 40 clones interacted with AT1R as judged by growth on selective media. Sequence analyses revealed that 21 of the 40 recovered clones encoded GABARAP or the related protein GABARAPL1. Both GABARAP and GABARAPL1 (data not shown for the latter) interacted specifically with AT1RCT; interactions with heme oxygenase-1 and biliverdin reductase were not observed in yeast (Figure 1). GABARAP is 87% identical to GABARAPL1 at the amino acid sequence level, whereas GABARAP: GABARAPL2 are 58% identical and GABARAPL1: GABARAPL2 are 61% identical; GABARAPL2 was not recovered from this screening.
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GABARAP and GABARAPL1 encode very similar proteins belonging to a new MAP family and possess amino-terminal tubulin-binding domains (residues 1 to 27).13 Not surprisingly, all GABARAP(X) clones recovered from the Y2H screen encode partial proteins (see Figure I in the online data supplement for more information).
GABARAP Interacts With the AT1R C-Terminal Cytoplasmic Sequence in Mammalian Cells
The AT1R:GABARAP interaction observed in yeast was initially verified in mammalian cells using GST pull-down assays (Figure 2A). CHO-K1 cells were transiently transfected with pCMV/HA/GABARAP, pGST/AT1RCT, or both plasmids, and cell extracts were prepared at 24 hours posttransfection. Extracts were applied to glutathione columns, washed, eluted, and electrophoresed on denaturing gels. Filters were probed with anti-hemagglutinin (HA) antibodies to confirm that AT1RCT specifically binds GABARAP. HA-GABARAP clearly associates with GST-AT1RCT and is coeluted from the glutathione column.
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GABARAP Coimmunoprecipitates With AT1R in Mammalian Cell Extracts
CHO-K1 cells were transfected with pFlag/AT1R and pCMV/HA/GABARAP (or empty vector [–]) or pCMV/HA/GABARAPL1 (or empty vector [–]) (Figure 2B). Twenty-four hours posttransfection, cell extracts were prepared and immunoprecipitated with anti-Flag antibody or preimmune serum (control) and protein A agarose. The immunoblot was probed with anti-HA antibody. The results indicate that the Flag/AT1R interacts with GABARAP and with GABARAPL1 in CHO-K1 cells.
In parallel studies, the intermolecular complexes of HA/GABARAP:Flag/AT1R and HA/GABARAPL1:Flag/AT1R were immunoprecipitated with resin-bound anti-Flag antibodies. Resulting immunoblots show that the AT1R complexes with GABARAP (Figure 2C) and GABARAPL1 (Figure 2D) in mammalian cells.
GABARAP Interacts Directly With AT1RCT
GST pull-down assays using purified recombinant proteins were carried out to determine whether GABARAP binds directly to AT1RCT. As shown in Figure 2E, recombinant GABARAP is eluted from resin charged with the GST-AT1RCT fusion but not with GST alone (compare lanes 1 and 3), providing evidence for direct interaction between these proteins.
Bioluminescence Resonance Energy Transfer for GABARAP:AT1R Interactions
Bioluminescence resonance energy transfer (BRET) assays (Figure 3) indicate a significant intermolecular interaction between GABARAP and AT1R (pGFP2/GABARAP+pAT1R/ RLuc), P<0.001 versus pAT1R/RLuc alone. (Note that all values have been corrected by subtracting the mock-transfected cell background at each emission wavelength, a function of the BRET program.) General negative controls include pAT1R/RLuc transfected alone and cotransfected empty vectors (pGFP2-C1+pRLuc-N1). Experiment-specific negative controls include mouse MAP1 light chain 3 (MAP1 LC3) as a GABARAP analog and the endothelin type A GPCR (ETR-A) as an AT1R correlate. The endothelin receptor is from the same GPCR family as the AT1R (Family 1, subgroup 1A) and, therefore, serves to show that GABARAP does not promiscuously interact with GPCRs. MAP1 LC3 is a MAP that binds to both MAP1 and MAP2 and, in this case, demonstrates that binding to the Ang receptor C terminus is not a general property of MAPs. Neither the general nor specific negative controls demonstrate significant BRET. The positive control, a commercial vector, pGFP2-RLuc, that encodes a cytomegalovirus (CMV)-regulated fusion protein of Renilla luciferase with GFP2, has been shown to be expressed at high levels in a variety of cells and, indeed, shows very high energy transfer in our assay (P<0.001 versus pAT1R/RLuc-transfected; P<0.001 versus pGFP2/GABARAP+pAT1R/Rluc). The BRET2 assays verify and support our Y2H results, GST pull-down assays, and coimmunoprecipitation results. See the online data supplement (expanded Results section) for more information.
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Influence of GABARAP Overexpression on AT1R Accumulation and Plasma Membrane Presentation
PC-12 neural cells were plated, treated with NGF (7S, 100 ng/mL) for 4 days (to enhance the neuronal differentiation),14,15 and then transfected with pAT1R/EYFP or pECFP/GABARAP+pAT1R/EYFP. Cells were evaluated at 24 and 48 hours posttransfection for cell surface expression of AT1R/EYFP using 3D deconvolution microscopy. Under our transfection conditions, AT1R/EYFP, as reported in our previous studies, is observed at the plasma membrane but is found predominantly in the secretory pathway (endoplasmic reticulum, Golgi, vesicles) (Figure 4A1 and 4A2). By 48 hours posttransfection, GABARAP overexpression increased AT1R cell surface expression 6.74-fold when standardized to the level of intracellular expression (P<0.005, n=3, 100 transfected cells per experiment) (Figure 4B through 4D). See the online data supplement (expanded Results section) for more details.
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Radioligand Binding Assays
CHO-K1 cells, which do not to express detectable Ang receptor,16–18 were stably transfected with AT1R/EYFP to obtain a working cell line with a defined Ang receptor. One high-level fluorescent clonal isolate was subsequently transfected with either pECFP/GABARAP or pECFP-C1 (control) and mixed (nonclonal) stable double transfectants were selected. In this manner, the AT1R uninduced baseline expression in both double-transfectant lines should be equal. Our studies show that expression of ECFP/GABARAP increases total binding 3.6-fold over the ECFP-C1 control (Figure 5). This increase is sensitive the AT1R blocker losartan but not to the AT2R blocker PD123319. GABARAP coexpression clearly increases not only the cell surface manifestation of AT1R/EYFP (Figure 4) but also the number of plasma membrane-associated Ang II binding sites.
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Immunoblots
By Western blot analysis, we observed a 7.7-fold AT1R protein increase (n=3, P<0.001) in COS-7 cells transfected with pCMV/myc/AT1R+pCMV/HA/GABARAP as compared with pCMV/myc/AT1R with either pCMV/Flag/SNAPAP (control) or control unmodified vectors (Figure 6). Therefore, GABARAP not only alters the distribution but also increases the steady-state AT1R protein level.
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SNAPAP was included as a negative control in this study. We isolated SNAPAP as a potential AT1R binding partner in an initial screen of the Y2H library, but we were subsequently unable to confirm it by affinity pull-down or by coimmunoprecipitation assays. We later learned that SNAPAP (also called SNAPIN) is frequently isolated as a false-positive in initial Y2H assays performed in 2-hybrid service facilities (personal communication, Chandra Tucker, Duke Yeast Model Systems Genomics Group).
GABARAP Effects on Extracellular Signal-Regulated Kinase Activation
Ang II:AT1R-mediated extracellular signal-regulated kinase (ERK)1/2 phosphorylation stimulation is known to occur in many cell types and through several mechanisms.19–21 Because our imaging data and radioligand binding assays suggest that GABARAP increases plasma membrane accumulation of the AT1R, as well as ligand association, we asked whether GABARAP might also (indirectly through increasing plasma membrane accumulation of receptor) stimulate ERK phosphorylation (Figure 7A and 7B). Our data show that Ang II treatment of pAT1R/EYFP stably transfected CHO-K1 cells increases the ratio of phosphorylated to unphosphorylated ERK1/2 levels 2.7-fold over vehicle treatment (n=3, P<0.005). In comparison, Ang II treatment of pECFP/GABARAP, pAT1R/EYFP double-transfectants increases the ratio 5.5-fold (n=3, P<0.005). GABARAP overexpression, therefore, significantly augments accumulation of phospho-ERK1/2 levels (n=3, P<0.005), consistent with greater plasma membrane expression of functional AT1R in double-transfectants.
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Proliferation Assays
Because Ang II has been shown to mediate proliferation of many cell types through the AT1R19,22,23 and our data indicate that GABARAP increases cell surface expression of the AT1R, we measured and compared growth of CHO-K1-pAT1R/EYFP-pECFP/GABARAP versus CHO-K1-pAT1R/EYFP-pECFP in the presence of Ang II, losartan (AT1R blocker), and/or PD123319 (AT2R blocker) as in Cook et al.19 Ang II increases both 5-bromodeoxyuridine incorporation into DNA and cell counts (3.5- and 3.3-fold, respectively) in the AT1R engineered CHO-K1 cell line in a manner that is sensitive to losartan but refractory to PD123319 (Figure 7C and 7D). Furthermore, Ang II promotes cell proliferation to a greater extent (approximately 2-fold greater) in the AT1R/GABARAP as compared with the AT1R recombinant cell line, consistent with higher plasma membrane expression of functional AT1R in these cells.
Small Interfering RNA–Mediated Knockdown of GABARAP Reduces Cell Surface Expression of Recombinant AT1R/EYFP
The ability of GABARAP complementary small interfering (si)RNA oligonucleotides to reduce GABARAP protein was tested using hybrid complexes (siGABARAP-1, siGABARAP-2, and siGABARAP-3) made complementary to 3 phylogenetically conserved regions of the mRNA. CHO-K1 cells stably transfected with pECFP/GABARAP and pDsRed2-Nuc were transiently transfected with siRNA duplexes. Negative control (Silencer #1 from Ambion), and scrambled siGABARAPC-1 negative control caused no diminution in ECFP/GABARAP compared with mock-transfected cells (Figure 8, I). However, siGABARAP-1, siGABARAP-2, and siGABARAP-3 duplex RNAs all significantly reduced ECFP/GABARAP accumulation, as determined by both deconvolution image analysis (85% to 93% reduction, P<0.005 versus scrambled negative control [siGABARAPC-1], n=3 experiments/hybrid complex, 100 cells evaluated/experiment) (Figure 8, I) and Western blot analyses (Figure 8, II) (standardized 5.3-fold reduction of ECFP/GABARAP levels in siRNA-treated cells [P<0.001 versus mock, n=3 experiments]). Neither negative control RNAs nor experimental siRNAs significantly affected DsRed2-Nuc nuclear fluorescence.
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The GABARAP siRNA duplexes were subsequently applied to CHO-K1 cells stably transfected with pAT1R/EYFP and pDsRed2-Nuc to determine whether a reduction in native GABARAP might also diminish fluorescent AT1R plasma membrane accumulation. GABARAP siRNAs reduced AT1R/EYFP steady-state levels an average of 43% (P<0.005 versus scrambled negative control [siGABARAPC-1], n=3 experiments/hybrid complex, 150 cells evaluated/experiment) (Figure 8, III) and with 84% reduction in plasma membrane-associated yellow fluorescence (P<0.01 versus scrambled negative control; SlideBook 4.2 software, "Masks" to quantify regions of interest). Immunoblot analyses were also performed to verify the quantitative image data (Figure 8, IV). GABARAP siRNA transfection (for any of the 3 hybrid complexes) reduced AT1R/EYFP expression an average of 4.2-fold as compared with transfection with scrambled control siRNA (P<0.005, n=3 experiments).
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Discussion |
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In this report, we show, by 3D deconvolution imaging and radioligand binding, that GABARAP, a MAP involved in GABAA receptor trafficking, increases plasma membrane accumulation of the AT1R. We further show that the increase in cell surface AT1R correlates, in response to Ang II, with increased phospho-ERK1/2 accumulation and enhanced proliferation. We further demonstrate association of these 2 proteins by yeast complementation, GST affinity pull-down, coimmunoprecipitation assays, and BRET assays. In addition, GABARAP-targeted siRNAs effectively reduce cell surface accumulation of fluorescent AT1R, demonstrating the importance of endogenous GABARAP for efficient plasma membrane-directed transport.
Using a similar approach, Dzau and colleagues have discovered that the AT1R binds to a transmembrane protein, ATRAP (Ang II type 1 receptor-associated protein), a 162-aa 3-transmembrane protein that modulates Ang II signaling.24,25 ATRAP appears to generally downregulate Ang II–mediated AT1R function, reducing inositol lipid levels, decreasing Ang II–mediated c-fos transcription, and reducing cell proliferation. ATRAP was isolated from a mouse kidney yeast library using the AT1AR amino acids 297 to 359 as bait. In contrast, we did not recover ATRAP from a mouse brain yeast library. ATRAP mRNA does appear to be ubiquitous, but it is apparently present at very low levels in brain compared with the levels in kidney, testis, or heart,25 perhaps accounting for our failure to isolate this message. ATRAP binds to the AT1R through the ATRAP C-terminal amino acids 110 to 122. AT1R binds to ATRAP through the AT1R terminal 20 amino acids (339 to 359).
By further example, using rat AT1AR amino acids 295 to 359 as bait to screen a mouse 10 day embryo library, Guo et al isolated ARAP1 (type I Ang II receptor associated protein 1), a ubiquitous 493-aa protein that interacts (in rather broad terms) with residues 319 to 359 of the AT1R.26 ARAP appears to enhance receptor recycling to the plasma membrane and receptor resensitization to a second Ang II stimulus. Transgenic mice overexpressing ARAP1 in kidney proximal tubule cells demonstrate high blood pressure and kidney hypertrophy.12 Therefore, both ATRAP and ARAP1 bind to the distal portion of the AT1AR C terminus and mediate quite different effects; ATRAP attenuates the Ang II–mediated AT1R downstream effects, whereas ARAP1 potentiates Ang II effects by upregulating the receptor level. Both of these also appear to interact with the distal portion of the AT1R C terminus. In contrast, our preliminary studies (data not shown) suggest that the binding site for GABARAP is in the membrane proximal region of the AT1R cytoplasmic C terminus (proximal to residue 316). Both ARAP1 and GABARAP interactions with AT1R appear to serve, in a similar capacity, to upregulate the renin–Ang system.
GABARAP family members appear to be highly connected with binding partners that include NSF (N-ethylmaleimide–sensitive factor),27,28 tubulin (and microtubules),4,29,30 transferrin receptor,31 GABAAR,32–34 AT1R, DDX 47 (an RNA helicase),35 GRIP1 (an adapter and steering protein),36 gephyrin,37,38 ULK1 (a neuronal serine/threonine kinase involved in axonal elongation),39 and p130 (inositol triphosphate binding protein).40 GABARAP has, in fact, been referred to as a "multifunctional adapter molecule" because of the myriad of binding partners associated with it.30 The fact that GABARAP is involved in trafficking of both the pentameric ionotropic GABAAR and the 7-transmembrane GPCR AT1R suggests that these receptors could be cotransported/coregulated and that conditions that favor upregulation of one may also favor upregulation of the other. This remains to be tested. GABARAP is a ubiquitous protein though it is primarily known for its role in trafficking of the GABAAR in the central nervous system. Our studies indicate that interaction of AT1R with GABARAP occurs in nonneuronal cells, although the relative importance of GABARAP for AT1R trafficking in neural and nonneural tissue is yet unknown.
Two primary motor proteins control microtubule-based protein transport. Anterograde trafficking (toward the plasma membrane) involves the motor protein kinesin, whereas retrograde movement involves dynein. Kinesin is an ATP-binding protein that "walks" along the length of the microtubule with ATP hydrolysis occurring at each successive 8-nm step. In contrast, GABARAP is not a nucleotide-binding motor protein and does not directly convey the AT1R- or GABAAR-containing vesicles to the plasma membrane. Nonetheless, GABARAP increases the steady-state level of these proteins at the plasma membrane. How might GABARAP enhance plasma membrane accumulation of proteins like GABAAR and AT1R?
For the AT1R, this appears to be, in part, attributable to accumulation of a higher level of receptor as compared with total cell protein in the presence of GABARAP. However, in addition, the trafficking process is altered as established by the change in the distribution of fluorescent AT1R when expressed with GABARAP. The relative AT1R cell surface-to-intracellular (secretory pathway) distribution is altered in PC-12 cells overexpressing GABARAP (a cytoplasmic protein). Clearly, GABARAP overexpression causes relatively more AT1R accumulation at the plasma membrane compared with the internal compartments. This observation, coupled with the knowledge that GABARAP binds tubulin and microtubules, suggests that GABARAP enhances the transport process. Because GABARAP is not a motor protein and does not actively move vesicular cargo to the plasma membrane, how might it enhance transport? One possibility is that GABARAP increases the processivity of plasma membrane trafficking. A kinesin molecule, on average, completes a 1-µm run length before dissociating from a microtubule.41 Often, cargos must be moved considerably further than 1 µm to reach their destination. Therefore, vesicular cargos must be passed from one to another microtubule-engaged kinesin complex to continue processive movement to the plasma membrane. GABARAP could function, therefore, as an accessory protein, binding both vesicle-associated AT1R and microtubules, so that AT1R containing vesicles are retained on the microtubules, on motor protein dissociation, until the vesicle can be recovered by a second kinesin complex. In this model, GABARAP would bind both vesicle-associated AT1R and a microtubule and act as a "rear wheel" stabilizing the kinesin–vesicle complex on the microtubule as it moves the cargo forward. In fact, there does exist evidence to suggest that motors may use secondary binding sites to aid in processivity.42
Given the central role of the AT1R in blood pressure homeostasis and cardiovascular disease, regulation of proteins that are involved in trafficking of this receptor and mechanisms by which these proteins interact with the receptor and modify its accumulation and function are of critical importance. We are the first to report an interaction between the AT1 receptor and the trafficking protein GABARAP. This novel finding introduces new areas of investigation in the renin–Ang system and in basic mechanisms of Ang II–mediated growth and blood pressure control and offers a new potential therapeutic target for pharmacological intervention.
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Acknowledgments |
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Sources of Funding
This work was supported by the Ochsner Clinic Foundation and NIH/National Heart, Lung, and Blood Institute grant HL072795.
Disclosures
None.
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Footnotes |
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Original received October 22, 2007; resubmission received April 8, 2008; revised resubmission received May 9, 2008; accepted May 12, 2008.
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References |
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