A specific role of AGS3 in the surface expression of plasma membrane proteins

Abstract

Activator of G protein signaling 3 (AGS3), originally identified in a functional screen for mammalian proteins that activate heterotrimeric G protein signaling, is known to be involved in drug-seeking behavior and is up-regulated during cocaine withdrawal in animal models. These observations indicate a potential role for AGS3 in the formation or maintenance of neural plasticity. We have found that the overexpression of AGS3 alters the surface-to-total ratios of a subset of heterologously expressed plasma membrane receptors and channels. Further analysis of the endocytic trafficking of one such protein by a biotin-based internalization assay suggests that overexpression of AGS3 moderately affects the internalization or recycling of surface proteins. Moreover, AGS3 overexpression and siRNA-mediated knockdown of AGS3 both result in the dispersal of two endogenously expressed trans-Golgi network (TGN)-associated cargo proteins without influencing those in the cis- or medial-Golgi compartments. Finally, adding a TGN-localization signal to a CD4-derived reporter renders the trafficking of fusion protein sensitive to AGS3. Taken together, our data support a model wherein AGS3 modulates the protein trafficking along the TGN/plasma membrane/endosome loop.

Fig. 1.

The effects of overexpressing AGS3 or mPins on the surface and total protein levels of receptors and channels. (A) An antibody against AGS3 (CalBiochem) recognized a 74-kDa band from HeLa and COS7 lysates. Lysates from COS7 cells overexpressing an HA-tagged AGS3 and PC12 cells were included as positive controls (14). (B–F) Extracellular HA-tagged receptors or channels were cotransfected into COS7 cells with an equal amount of empty pcDNA3 vector (as a control) or pcDNA3 expressing AGS3. (B) Total protein levels were determined by a quantitative Western blot analysis using the Li-Cor Odyssey Infrared Imaging System. (C) Surface proteins levels were determined by the previously described chemiluminescence assay (19). (D) The surface-to-total ratio of Kir2.1 is increased by ≈42% in cells overexpressing AGS3. (E) A similar increase (≈36%) of the surface-to-total ratio of Kir2.1 was seen with a complementary biotinylation approach. 1x and 1y are arbitrary units and represent the total and surface levels of control samples, respectively. GAPDH was probed to assure the plasma membrane integrity during biotinylation. (F) Unlike AGS3, AGS3S and mPins both fail to significantly enhance the surface expression of Kir2.1. The biotinylation experiments were repeated twice and similar results were obtained. The values shown in the other graphs are the average of at least six independent experiments.

Fig. 2.

Effects of AGS3 are independent of the PDZ-binding motifs of Kir2 channel proteins. Although AGS3 greatly increases the surface expression of Kir2.1, it does not significantly alter that of Kir2.2 or Kir2.3. Moreover, deletion of the PDZ-binding motif in Kir2.1Δ3 has no impact on the ability of AGS3 to stimulate the surface level of Kir2.1. Surface chemiluminescence measurements were conducted as described in Fig. 1. The values shown are the average of at least six independent experiments.

Fig. 3.

Overexpression of AGS3 modestly affects the internalization or recycling of Kir2.1 at later, but not earlier, time points when compared with control. Surface proteins of COS7 cells overexpressing Kir2.1 and either AGS3 or pcDNA3 (control) were labeled by using the membrane-impermeant Sulfo-NHS-SS-Biotin (Pierce) and allowed to internalize for a period of 0, 5, 10, or 20 min. Remaining surface biotin was cleaved by using a membrane-impermeant reducing agent, mercaptoethanesulfonic acid (MESNA, 50 mM). Samples left untreated with MESNA at time 0 represent the total surface Kir2.1 protein. Proteins retaining their biotin tags after the MESNA treatment thus represented surface proteins that had become internalized but not yet recycled back to the plasma membrane. (A) Amounts of Kir2.1 remaining internalized after 0, 5, 10, and 20 min were normalized to total surface expression. Values shown are the average of four separate sets of Western blots. (B) A representative set of Western blots of the Kir2.1 protein internalization assay is shown.

Fig. 4.

Inhibition of clathrin-mediated internalization of Kir2.1 does not prevent the stimulation of channel surface expression by AG53 overexpression. (A) Surface expression of Kir2.1 was determined by chemiluminescence assay as in Fig. 1. Expression of AP180C efficiently inhibits the internalization of Kir2.1 (comparing Kir2.1 to Kir2.1+AP180C); however, it does not influence the AGS3-dependent increase of surface Kir2.1 (≈40% in either case, comparing Kir2.1+AGS3 with Kir2.1+AGS3+AP180C). Equal amounts of Kir2.1 were used for transfection in each experiment. Because a smaller amount of AGS3 was used for each transfection, the effects of AGS3 on the surface level of Kir2.1 appeared to be less compared with that in Figs. 1 or 2. (B) Exogenous biotinylated transferrin receptors are internalized normally and concentrated at the perinuclear recycling endosomes (indicated by arrows) in COS7 cells overexpressing AGS3.

Fig. 5.

Overexpression of AGS3 disrupts the localization of two endogenous TGN cargo proteins without affecting those of cis-Golgi/cis-Golgi network and medial-Golgi proteins. (A) COS7 cells overexpressing AGS3 were counted (n > 200, only cells highly overexpressing AG53 were counted) based on whether the staining of Golgi marker proteins had predominantly perinuclear or diffuse staining. (B) The localization of p115 is not significantly different in those cells highly overexpressing AGS3, whereas localization of the TGN cargo proteins TGN46 and CD-MPR are dramatically altered in cells with high levels of AGS3. Cells overexpressing and not overexpressing AGS3 were captured in the same field for comparison. (Scale bars: 20 μm.)

Fig. 6.

Similar to the overexpression of AGS3, depletion of AGS3 also leads to the specific dispersal of endogenous TGN cargo proteins. (A) The AGS3 siRNA used in this study efficiently knocked down the AGS3 protein level by 80% (Western blot quantified by the Li-Cor Odyssey Infrared Imaging System) without influencing that of GAPDH. (B) HeLa cells were counted (n > 200) based on whether the subcellular distributions of marker proteins were normal or diffuse. (C) p115 localization changes very little when cells are treated with the AGS3 siRNA, whereas the staining of the TGN cargo proteins, CD-MPR and TGN46, is much more diffuse when compared with control cells. (Scale bars: 20 μm.)

Fig. 7.

Elevated AGS3 greatly increases the surface expression of two TGN-enriched CD4-derived reporters. (A) Whereas CD4ΔC is efficiently expressed on the plasma membrane, both CD4ΔC-Tcd and CD4ΔC-Fcd primarily reside at the TGN. An anti-TGN46 antibody was used to label the TGN. (B) Surface chemiluminescence measurements of CD4ΔC, CD4ΔC-Fcd, and CD4ΔC-Tcd plasma surface expression were performed as described in Fig. 1. (C) A complementary surface biotinylation assay was used to determine surface levels of CD4ΔC and CD4ΔC-Fcd. 1x and 1y are arbitrary units and represent the total and surface levels of control samples (i.e., cells transfected with pcDNA3 only), respectively. AGS3 overexpression caused an increase in the CD4ΔC and CD4ΔC-Fcd total protein levels compared with controls (18% and 56%, respectively) and an increase in the surface protein levels of the two by 31% and 145%, respectively. Thus, the surface-to-total ratio of CD4ΔC-Fcd was stimulated by AGS3 to a much higher extent compared with that of CD4ΔC.