Endosomes internalize integral plasma membrane and membrane-associated proteins, which subsequently can be recycled back to the plasma membranes, transported to other membranes and/or delivered to lysosomes or proteasomes for degradation . For many proteins endocytosis is simply a mechanism for transportation from one location to another, however internalized receptors and other proteins may function and be regulated in unique ways [2, 5, 7, 9, 36] and thus endosomes are not simply cargo vessels. For example, internalized β2 adrenergic receptor and β-arrestins contribute to formation of signalling complexes and may initiate unique signalling pathways [2, 8]. Signal transduction from circulating hormones is a critical function of hepatocyte plasma membranes. Although intracellular signalling from insulin receptors has been well characterized in liver [2, 5–8, 36], little is known regarding the role of endocytosis in signalling from GPCRs involved in cAMP-mediated signal transduction [2, 8, 11], especially as regards the physical location of heterotrimeric G protein subunits that couple to GPCRs in liver.
Further, previous studies suggested that signalling through Gs and Gi proteins may, in turn, alter endocytosis and endosome trafficking [11, 16, 17, 37]. Therefore a systematic study was undertaken to determine whether heterotrimeric G protein subunits were associated with hepatocyte endosomes as the first step in assessing whether internalized GPCR, like insulin and EGF receptors, can generate signals from endosomes.
Three different approaches were used in the present study. First, Western blotting was performed with purified endocytic vesicles and plasma membranes. Although the principal goal was to identify heterotrimeric G protein subunits on intracellular endocytic vesicles, a second goal was to compare/contrast the rank order of G proteins with other marker proteins as evidence for internalization and recycling and against contamination of vesicles by plasma membranes.
A plasma membrane preparation was selected that is considered free of intracellular organelles, although exhibiting 8% contamination of BLM with CM . However, CM proteins may traffic from Golgi-to-BLM-to-CM  and therefore be found on BLM as well. As few methods are available to purify rat liver endosomes, clathrin-coated vesicles, the earliest stage of receptor-mediated endocytosis, also exist in Golgi, and heterotrimeric G proteins may be internalized in non-coated vesicles , a method was chosen that allows simultaneous preparation of three different types of endocytic vesicles, representing different steps in receptor-mediated endocytosis and destinations for probes of fluid-phase endocytosis [18–20]. These vesicles have been used to study liver receptor-mediated and fluid-phase endocytosis, endosome ion transport and association of proteins with endosomes [18–24, 31]. During receptor-mediated endocytosis recycling receptors are found in these vesicles in the order RRC>CURL>MVB [17, 19, 23, 24, 30]. Purified secondary lysosomes  were studied also as internalized proteins destined for degradation are transferred from early endosomes to CURL, to MVB and on to lysosomes. Such proteins are enriched in the order MVB>CURL>RRC [18, 19, 21, 23, 24]. Heterotrimeric G protein subunits may be degraded primarily via ubiquination and proteasomes , thus little likely enters lysosomes.
By Western blotting three Gα and one Gβ subunit were found on CM and BLM, qualitatively similar to the results of others using different membrane preparative methods [3, 4]. These G protein subunits were found in the order CM ≥BLM>>>RRC>CURL≥MVB≥lysosomes (Figure 1), an order expected for proteins endocytosed and then recycled back to the plasma membrane and similar to the order identified for Trf-R (Figure 2) [23, 24, 30]. Others identified a similar order for other signalling molecules including EGF receptors, Ras, Raf-1, MAPK kinase, MAPK and phospho-MAPK [6, 9, 19, 24]. The heterotrimeric G protein subunits likely were attached to the cytoplasmic surface of endosomes, a position that allows signal transduction. However MVB internalize their own membranes as topographically inverted internal vesicles , thus some of these G proteins may be delivered to lysosomes as internalized cargo.
These results agree with those of others who identified Gsα and generic Giα on RRC, CURL and MVB in the same order (Figure 1) [24, 31]. Further we also identified the Gs effector AC on CM, BLM and endocytic vesicles (Figure 5). AC activity, but not protein content, was previously demonstrated on liver BLM and CM . Collectively these findings confirm and extend the observations of others and support the hypothesis that heterotrimeric G proteins may participate in intracellular signal transduction.
Western blot methods critically depend on the purity of the samples. Given the large amount of Gsα, Giα1,2, Giα3 and Gβ on CM and BLM (Figure 1), contamination of endosomes with up to 5–30% CM or BLM might explain the results. This high a contamination seems unlikely, but merits serious consideration. Therefore the distribution, in vesicles, of other plasma membrane proteins was examined. If contamination was the explanation, all should have exhibited the same pattern (rank order). Gsα, Giα1,2, Giα3 and Gβ were found in the same order identified for recycling receptors and the α1 subunit of Na, K-ATPase (Figure 2). By contrast, MDR-1 and MRP-2, which likely traffic from Golgi-to-BLM-to-CM and are subsequently endocytosed from CM and degraded in lysosomes [32–34, 39] were detected in the order MVB > CURL~RRC, similar to the order of endocytosed proteins degraded in lysosomes [18, 19, 21, 23, 24].
We also examined distribution of rab 5 and rab 4, markers of early and recycling endosomes [29, 30], respectively, that are also associated with plasma membranes. RRC as a combination of early, recycling and transcytotic vesicles  are expected to exhibit rab 4 and rab 5 while receptor-containing appendages of CURL  might exhibit rab 4. Others found both rabs in the order RRC≥CURL>MVB [22–24]. However, using the same antibodies we were unable to identify rabs in our endosomes (Figure 3), possibly as our x-ray films may not have been exposed as long due to large amounts of rabs on plasma membrane samples. Conversely, the absence of detectable rab 5 and rab 4 in our endosomes suggests that contamination of endosomes by CM or BLM must be small.
Quantitative study of the distribution of Na, K-ATPase α1 and β1 subunits also suggested little contamination of endocytic vesicles by plasma membranes. As previously reported by others for different membrane preparations , both subunits were detected in CM and BLM with β1 in larger amounts in BLM. In intracellular vesicles α1 was detected in the order RRC>CURL>MVB>lysosomes, the same found for Gsα, Giα1,2, and Gβ (Figure 1) and recycling receptors, which suggests α1 is internalized and recycled. Others identified α1 only in RRC , suggesting differences in techniques or antibody lot. Although β1 could not be identified in our vesicles (Figure 2), β1 is found in rat liver early endosomes . Since the antibody employed was capable of demonstrating β1 in even small amounts of BLM (Figure 2), the lack of β1 in RRC/CURL/MVB/lysosomes is likely due to rapid loss of β1 after endocytosis by ubiquination and proteasome degradation , resulting in high α/β ratio .
A second method for demonstrating colocalization of proteins and intracellular vesicles is the pattern of distribution on density gradients. We used PNS to minimize bias from selective loss of any cellular organelles and to complement the experiments performed using highly purified plasma membranes and endocytic vesicles. Gsα, Giα3 and Gβ were readily detectable throughout most of the gradient, paralleling detection of endocytosed FITC-dextran, even in regions of the gradient where markers of plasma membranes and Golgi vesicles were minimally detected (Figure 4). Giα1,2, however, was identified principally in regions where the bulk of endosomes and the trans-Golgi network (identified by TGN-38) were found, consistent with reports in other cell types [14, 15]. Clean separation of plasma membranes from low density intracellular organelles on such gradients is not possible , therefore the results shown here do not constitute conclusive proof. However these findings are consistent with presence of at least some heterotrimeric G protein subunits on endocytic vesicles.
The third approach was direct visualization of Gsα, Giα1,2, Giα3 and Gβ by confocal microscopy of liver in which endosomes were labelled by the endocytosed fluid phase marker Texas red-dextran . Major advantages of confocal microscopy include verification that these proteins are in hepatocytes and the ability to identify co-localization with endocytosed probes. We previously showed that fluorescent dextrans endocytosed from blood at the BLM first appear in punctate vesicles just under the BLM and then rapidly traffic, presumably on microtubules, to the pericanalicular region where endosomes, lysosomes and elements of the Golgi Apparatus are found . Vesicles endocytosed from the CM  are not labelled with Texas red-dextran. In CTX-treated livers, mistrafficked early and late endosomes form perinuclear clusters, spatially separated from BLM and CM .
Gsα, Giα3 and Gβ were identified on CM and BLM, consistent with limited previous studies of mouse liver  and with Western blots (Figure 1) [3, 26]. In addition punctate staining was identified adjacent to and under CM and BLM, a pattern interpreted as indicating protein in, or attached to, vesicles, including endosomes. This punctate staining was visible near CM after two minutes of dextran endocytosis when no dextran-loaded endosomes were found there, ruling out bleed-through from the Texas red signal (Figures 6B,7F,8B). Punctate staining also was found in perinuclear regions in CTX-treated livers (Figures 6F,8H), where staining of plasma membranes could not create artifacts. These findings provide strong support for our hypothesis that heterotrimeric G proteins are indeed located on endocytic vesicles.
Giα3 was observed also faintly distributed over the cytoplasm, although not in a pattern specific to any known organelle. Others have identified Giα3 by immunofluorescence on plasma membranes, nuclei, Golgi Apparatus and in subapical compartments in a variety of cell types [14, 15, 43, 44].
We were unable to localize Giα1,2 to membranes or endocytic vesicles due to the intense nuclear and diffuse cytoplasmic staining. This finding differs from the ready detection of Giα1,2 on membranes and endosomes by Western blotting (Figure 1), but may reflect inadequacies of the antibody to detect the native protein in fixed tissue or the concurrent localization of this G protein to other organelles [14, 15]. Indeed others have identified Giα1,2 by immunofluorescence in many locations including cytosol, on intracellular organelles, nuclei, actin filaments and/or on plasma membranes [14, 15, 43, 44]. Thus no definitive conclusions can be drawn from the confocal microscopy studies regarding membrane localization of Giα1,2.