HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 42, 44101-44112, October 15, 2004

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 279/42/44101    most recent M405151200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hynes, T. R. Articles by Berlot, C. H. Search for Related Content PubMed PubMed Citation Articles by Hynes, T. R. Articles by Berlot, C. H. Social Bookmarking                      What's this?

Live Cell Imaging of G_s and the ₂-Adrenergic Receptor Demonstrates That Both _s and ₁₇ Internalize upon Stimulation and Exhibit Similar Trafficking Patterns That Differ from That of the ₂-Adrenergic Receptor^*

Thomas R. Hynes, Stacy M. Mervine, Evan A. Yost, Jonathan L. Sabo, and Catherine H. Berlot

From the Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2623

Received for publication, May 10, 2004 , and in revised form, July 1, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To visualize and investigate the regulation of the localization patterns of G_s and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. _s-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of _s, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of _s. Functional fluorescent ₁₇ dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to ₁ (YFP-N-₁) and a carboxyl-terminal YFP fragment to ₇ (YFP-C-₇). When expressed together, YFP-N-₁ and YFP-C-₇ produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing _s-CFP, YFP-N-₁/YFP-C-₇, and the ₂-adrenergic receptor (₂AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, _s-CFP and YFP-N-₁/YFP-C-₇ stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with ₂AR-labeled vesicles. Moreover, internalization of ₂AR-GFP, but not _s-CFP or YFP-N-₁/YFP-C-₇, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the G_s subunits and ₂AR utilize different internalization mechanisms. Subsequent trafficking of the G_s subunits and ₂AR also differed in that vesicles labeled with the G_s subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of _s and ₁₇ on these endosomes may indicate a means of recycling specific _s combinations to the plasma membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Multiple G protein signaling pathways operate in individual cells to maintain homeostasis and to bring about responses to external stimuli such as growth and differentiation. An important, but unresolved issue is how the specificity of these pathways is maintained among so much complexity. 23 G protein subunits, 5 subunits, and 12 subunits have been identified in mammals (1), which could give rise to more than 1300 combinations. However, inactivation of specific G protein subunits in vivo using antisense (2-6) and ribozyme (7, 8) strategies has demonstrated a remarkable specificity of interaction between receptors, combinations, and effectors. For instance, ribozyme-mediated suppression of ₇ in HEK-293 cells specifically reduces expression of ₁ and disrupts activation of G_s by -adrenergic and D₁ dopamine receptors, but not by prostaglandin E₁ and D₅ dopamine receptors (8, 9). Moreover, knockout of ₇ in the mouse results in behavioral changes and reductions in the level of _olf in the striatum (10).

One mechanism by which signaling specificity appears to be regulated is at the level of subcellular compartmentalization, which can facilitate or impair interactions between proteins expressed in the same cell (11, 12). However, in the case of protein complexes such as G_s, for which the localization patterns of the and subunits have been reported to change upon activation, it is not clear how specificity can be maintained. The G_s subunits associate with the plasma membrane as a result of fatty acid modifications and association with each other. Targeting of subunits to the plasma membrane requires association with prenylated subunits (13) and is facilitated by association with subunits (14). Similarly, _s attaches to the plasma membrane as a result of amino-terminal palmitoylation (15, 16) and association with (17). Activation of G_s results in depalmitoylation of _s (18), and studies using immunohistochemistry (19) and an _s-GFP¹ fusion protein (20) have demonstrated activation-dependent movement of _s from the plasma membrane to the cytoplasm. Activation-dependent changes in localization have not been imaged in cells, but subcellular fractionation indicated that redistributed from the plasma membrane to low density microsomes upon stimulation of -adrenergic receptors (21). In the face of these localization changes, it is not clear how specific _s combinations can be preserved throughout multiple signaling cycles.

To address this issue, we have performed real time imaging of a G_s heterotrimer, _s17, which mediates signaling from the ₂AR to adenylyl cyclase (7, 8), in isoproterenol-stimulated HEK-293 cells. _s was visualized using an internally tagged _s-CFP fusion protein that has comparable activity to that of _s, whereas ₁ and ₇ were imaged exclusively in the form of ₁₇ complexes using the strategy of BiFC (22). BiFC involves the production of a fluorescent signal by two nonfluorescent fragments of YFP when they are brought together by interactions between proteins fused to each fragment. When expressed together, fusion proteins consisting of an amino-terminal YFP fragment fused to ₁ and a carboxyl-terminal YFP fragment fused to ₇ produce a fluorescent signal that is not obtained with either subunit alone (23). Our results indicate that both _s and ₁₇ internalize upon activation, showing significant co-localization on intracellular vesicles that are distinct from ₂AR-labeled vesicles. Moreover, internalization of the ₂AR, but not _s or ₁₇, was inhibited by a dominant negative dynamin 1 mutant, indicating that the G protein subunits dissociate from the receptor upon activation, and utilize a different mechanism for internalization from that of the receptor. Compared with vesicles labeled with the ₂AR, vesicles labeled with _s and ₁₇ exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Overlap of _s and ₁₇ with each other on these Rab11-labeled endosomes may indicate a means of recycling specific _s combinations to the plasma membrane, because Rab11 regulates trafficking through the pericentriolar recycling endosome (24). This approach of simultaneously imaging the and components of heterotrimeric G proteins in live cells will have many applications in elucidating the roles of specific combinations in particular signaling pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Production of Fluorescent Fusion Proteins—_s-CFP in the expression vector pcDNAI/Amp (Invitrogen) was generated from the long splice variant of rat _s cDNA (25) containing the EE epitope (26, 27) and ECFP (Clontech) containing a substitution of His for Asn¹⁶⁴ (28). BamHI sites in the _s cDNA were removed and _s residues 73-84 were then replaced with Gly-Ser, adding a unique BamHI site. ECFP was amplified by a PCR in which BamHI sites and linker sequences (Ser-Gly-Gly-Gly-Gly-Ser) were added at each end and then subcloned into the BamHI site in _s, so that ECFP was placed between Gly⁷² and Asp⁸⁵ (Fig. 1A). _s-GFP (20) in the expression vector pcDNA3.1 (Invitrogen) was kindly provided by Mark Rasenick. Production of YFP-N-₁ and YFP-C-₇ in pcDNAI/Amp was described previously (23).

View larger version (50K): [in this window] [in a new window]   FIG. 1. Production of a functional s-CFP fusion. A, model of s-CFP. The structure of CFP, based on that of GFP (79), is green. The helical domain of s·GTPS (80) is pink and the GTPase domain is light blue. GTPS is yellow. The SGGGGS linkers between CFP and s are shown schematically in dark blue. B, s and s-CFP are expressed at similar levels in HEK-293 cell membranes. Cells were transfected with plasmid encoding s (lane 1), s-CFP (lane 2), or vector (pcDNAI/Amp) (lane 3). Similar results were obtained in two additional experiments. C, s-CFP and s stimulate cAMP formation to similar extents in response to receptor activation. HEK-293 cells were transfected with 0.2 µg of plasmid encoding the porcine 2a-adrenergic receptor (81). In addition, the cells were transfected with a total of 0.5 µg of plasmid consisting of either vector alone (pcDNAI/Amp) or the indicated amounts of plasmid encoding s (circles) or s-CFP (squares) plus varying amounts of vector to keep the total amount of transfected plasmid constant. cAMP was measured in the presence (filled symbols) or absence (open symbols) of 10 µM UK-14,304. Values represent the means of triplicate determinations ± S.D. from a single experiment. Similar results were obtained in two additional experiments. D, s-CFP stimulates cAMP formation in response to receptor activation to a greater extent than a previously described s-GFP construct (20). HEK-293 cells were transfected as described for C using plasmid encoding s-CFP (circles) or s-GFP (squares). cAMP was measured in the presence (filled symbols) or absence (open symbols) of 10 µM UK-14,304. Values represent the means of triplicate determinations ± S.D. from a single experiment. Similar results were obtained in two additional experiments.

pECFP-Mem and pEYFP-Mem, encoding fusions to the amino termini of ECFP and EYFP, respectively, of the amino-terminal 20 residues of GAP-43, which leads to targeting primarily to the plasma membrane (32, 33), were obtained from Clontech. The fusion proteins are referred to as CFP-Mem and YFP-Mem. pECFP-Endo and pEYFPEndo, encoding fusions of ECFP and EYFP, respectively, to the amino terminus of the human RhoB GTPase, which localizes to early endosomes (34) and multivesicular bodies, a prelysosomal compartment (35), were obtained from Clontech. The fusion proteins are referred to as CFP-RhoB and YFP-RhoB and vesicles labeled with these markers are referred to as RhoB-labeled endosomes.

Transient Expression and Assay for cAMP Accumulation—HEK-293 cells (ATCC, CRL-1573) (10⁶ per 60-mm dish) were transfected with plasmids as described in the legend to Fig. 1 using 10 µl of LipofectAMINE 2000 reagent (Invitrogen). The cells were labeled with [³H]adenine and intracellular cAMP levels were determined as described previously (36) in the presence of 1 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine in the presence or absence of 10 µM UK-14,304.

Membrane Preparations and Immunoblots—HEK-293 cells (12.5 x 10⁶ per 150-mm dish) were transfected with 75 µg of plasmid using DEAE dextran (37). 48 h after transfection, membranes were prepared as described (36). 25 µg of membrane proteins were resolved by SDS-polyacrylamide electrophoresis (10%), transferred to nitrocellulose, and probed with a monoclonal antibody to the EE epitope (26) to compare _s and _s-CFP or with a polyclonal antibody to GFP (A.v. Peptide Antibody, Clontech) to compare _s-CFP and _s-GFP. The antigen-antibody complexes were detected according to the ECL Western blotting protocol (Amersham Biosciences). Chemiluminescence was imaged using a Lumi-Imager (Roche Applied Science).

Imaging of Fluorescent Fusion Proteins—HEK-293 cells were plated at a density of 10⁵ cells per well on Lab-Tek II, 4-well chambered coverslips and transiently transfected using 0.25 µl of LipofectAMINE 2000 reagent. Plasmids were transfected using the following amounts: _s and _s-CFP, 0.15 µg; ₁, YFP-N-₁, ₇, and YFP-C-₇, 0.075 µg; ₂AR, ₂AR-GFP, and ₂AR-CFP, 0.05 µg; mRFP, CFP-Mem, YFPMem, CFP-RhoB, YFP-RhoB, CFP-Rab11, and YFP-Rab11, 0.0125 µg; Dynamin-1-K44A-mRFP, 0.3 µg. Cells were imaged 2 days after transfection at x63 using a Zeiss Axiovert 200 fluorescence microscope under the control of IPLab software (Scanalytics) as described (23). Using the motorized x-y-z stage, time course images of cells located at 5-6 positions in the well were collected simultaneously. Cells selected for imaging expressed all of the labeled proteins, had a clearly delineated plasma membrane, and had a region of cytoplasm that was generally free of vesicles or other structures. Individual exposure times were optimized for each cell and color channel. Images for each color channel and DIC were collected at each position in the well every 60 s. Following the second time point, a stimulus of 200 µl of 40 µM isoproterenol was added to the well, resulting in a final concentration of 10 µM, and images were collected for 30 min. For each experimental condition, cells were imaged from plates transfected on 3 different days.

Image Analysis—Time course images were analyzed using IPLab software (Scanalytics Inc.). Before analysis, instrument background was subtracted from the images and corrections were made for bleaching so that the average intensity of the image was constant throughout the time course (see Supplemental Material). The in-focus membrane and vesicle features visible in the images were superimposed on a varying background of diffuse intensity because of out of focus, as well as soluble labeled protein. The images were processed to remove the low resolution diffuse background to produce a high resolution image that was used to identify groups of adjacent pixels or "segments" that corresponded to cellular features such as the plasma membrane and intracellular vesicles (see Supplemental Material). To distinguish between signals in the plasma membrane and in the cytoplasm, a "border" centered on the plasma membrane, 6-10 pixels wide (0.6-1.0 µm), was drawn around the edge of the cell using a Cintiq pen based display screen (Wacom).

Changes in the plasma membrane intensity of labeled proteins were measured in cells co-expressing a membrane marker (YFP-Mem or CFP-Mem) that was used to segment membrane pixels and correct for intensity changes due to changes in cell shape. A segment of pixels covering a length of the plasma membrane was identified from the high resolution image of the membrane marker, using a portion of the border as a mask (Fig. 5). The average intensities of these pixels in the background- and bleach-corrected images of the labeled protein and membrane marker were determined. The membrane marker intensity values were normalized to a starting value of one and the labeled protein intensity values were divided by the normalized membrane marker values. This corrected for changes in cell shape during the time course, because the distribution of the membrane marker did not change in response to agonist stimulation. The corrected labeled protein intensities were normalized to a starting value of one and averaged with values from multiple cells. Images of unstimulated cells analyzed in this manner showed no response or drift in signal (Fig. 3A). Cells were designated as non-responders if the plasma membrane intensity of the labeled protein dropped less than 3% during the 30 min time course.

View larger version (78K): [in this window] [in a new window]   FIG. 5. Isoproterenol-stimulated internalization of 2AR-GFP, but not s-CFP or YFP-N-1/YFP-C-7, is inhibited by Dyn1(K44A)mRFP. Top rows, before stimulation; bottom rows, after stimulation. In cells transfected with Dyn1(K44A)-mRFP, expression was confirmed by its fluorescence in each of the analyzed cells. A and B, Dyn1(K44A)-mRFP blocks isoproterenol-stimulated internalization of 2AR-GFP. Cells were transfected with s, 1, 7, 2AR-GFP, CFP-Mem, and either mRFP (A) or Dyn1(K44A)-mRFP (B). C and D, Dyn1(K44A)-mRFP does not block isoproterenol-stimulated internalization of s-CFP. Cells were transfected with s-CFP, 1, 7, 2AR, YFP-Mem, and either mRFP (C) or Dyn1(K44A)-mRFP (D). E and F, Dyn1(K44A)-mRFP does not block isoproterenol-stimulated internalization of YFP-N-1/YFP-C-7. Cells were transfected with s, YFP-N-1, YFP-C-7, 2AR, CFP-Mem, and either mRFP (E) or Dyn1(K44A)-mRFP (F). In the segments image, the cell border is gray, the segmented plasma membrane is green, the portion of the plasma membrane segment used for analysis of intensity is red, and intracellular vesicles labeled with 2AR-GFP, s-CFP, or YFP-N-1/YFP-C-7 are yellow. Bars = 10 µm. Videos for A-F of this figure are available under Supplemental Materials. G-I, quantification of internalization responses of 2AR-GFP, s-CFP, and YFP-N-1/YFP-C-7 relative to the plasma membrane marker (CFP-Mem or YFP-Mem) in the presence and absence of Dyn1(K44A)-mRFP. The number of cells analyzed and the percentage of cells that exhibited detectable responses (see "Experimental Procedures") are listed below. G, values for 2AR-GFP in the absence of Dyn1(K44A)-mRFP (filled circles, 30 cells, 87% responded) and presence of Dyn1(K44A)-mRFP (open circles, 38 cells, 26% responded). H, values for s-CFP in the absence of Dyn1(K44A)-mRFP (filled circles, 22 cells, 73% responded) and the presence of Dyn1(K44A)-mRFP (open circles, 30 cells, 77% responded). I, values for YFP-N-1/YFP-C-7 in the absence of Dyn1(K44A)-mRFP (filled circles, 30 cells, 60% responded) and in the presence of Dyn1(K44A)-mRFP (open circles, 39 cells, 72% responded). Values represent mean ± S.E. YN indicates YFP-N and YC indicates YFP-C.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Quantification of stimulus-dependent changes in the subcellular localization patterns of s-CFP and YFP-N-1/YFP-C-7. In this and all subsequent figures, cells were stimulated with 10 µM isoproterenol immediately after time = 0. A, stimulus-dependent loss of s-CFP (filled circles, 16 cells) and YFP-N-1/YFP-C-7 (filled squares, 18 cells) intensity from the plasma membrane. In unstimulated cells, the intensities of s-CFP (open circles, 18 cells) and YFP-N-1/YFP-C-7 (open squares, 20 cells) in the plasma membrane did not change. Measurements of s-CFP were made in cells that co-expressed s-CFP, YFP-Mem, and unlabeled 1, 7, and 2AR. Measurements of YFP-N-1/YFP-C-7 were made in cells that co-expressed YFP-N-1/YFP-C-7, CFP-Mem, and unlabeled s and 2AR. Values are the mean ± S.E. B, stimulus-dependent increases in intensity of s-CFP (circles) and YFP-N-1/YFP-C-7 (squares) in the cytoplasm (excluding labeled vesicles and the nucleus) in cells expressing s-CFP, YFP-N-1/YFP-C-7, 2AR, and mRFP (cytoplasm marker). Values represent the mean ± S.E. of 22 cells. C, stimulus-dependent labeling of vesicles with s-CFP and YFP-N-1/YFP-C-7. Areas of vesicles labeled with s-CFP (circles), YFP-N-1/YFP-C-7 (squares), and both s-CFP and YFP-N-1/YFP-C-7 (triangles) are shown. Values represent the mean ± S.E. of the same 22 cells that were analyzed for cytoplasm responses in B.

View larger version (86K): [in this window] [in a new window]   FIG. 2. s-CFP and YFP-N-1/YFP C-7 internalize in response to stimulation with 10 µM isoproterenol and exhibit substantial co-localization on vesicles. HEK-293 cells were transfected with plasmids expressing s-CFP, YFP-N-1/YFP-C-7, 2AR, and mRFP (cytoplasm marker, not shown). Prior to stimulation, s-CFP and YFP-N-1/YFP-C-7 localized to the plasma membrane (top row), and following stimulation, initially appeared in the cytoplasm (second row, 3 min) and then localized to vesicles (third row, 24 min). In this and subsequent figures, colors used in the merge images are listed following the construct name. Analysis of vesicle segments revealed a high degree of overlap (in yellow) of s-CFP (red) and YFP-N-1/YFP-C-7 (green) vesicles. The cytoplasm segment is shown in gray, and the cell border in blue. The video for this figure is available under Supplemental Materials. YN indicates YFP-N and YC indicates YFP-C. Bar = 10 µm.

View larger version (72K): [in this window] [in a new window]   FIG. 6. 2AR-GFP exhibits a greater amount of overlap with RhoB-labeled endosomes than do s-CFP and YFP-N-1/YFP-C-7. A, 2AR-GFP (red) exhibits significant overlap (yellow) with RhoB-labeled endosomes (green) after stimulation. Cells were transfected with s, 1, 7, 2AR-GFP, CFP-RhoB, and mRFP. Top row, before stimulation; bottom row, 25 min after stimulation. Cell border is blue. Bar = 10 µm. B, quantification of stimulus-dependent labeling of vesicles with 2AR-GFP and CFP-RhoB. Areas of vesicles labeled with 2AR-GFP (filled circles), CFP-RhoB (open circles), and both 2AR-GFP and CFP-RhoB (filled triangles) are shown. Values represent the mean ± S.E. from 22 cells. C, s-CFP (red) exhibits partial overlap (yellow) with RhoB-labeled endosomes (green) after stimulation. Cells were transfected with s-CFP, 1, 7, 2AR, YFP-RhoB, and mRFP. Top row, before stimulation; bottom row, 26 min after stimulation. Bar = 10 µm. D, quantification of stimulus-dependent labeling of vesicles with s-CFP and YFP-RhoB. Areas of vesicles labeled with s-CFP (filled circles), YFP-RhoB (open circles), and both s-CFP and YFP-RhoB (filled triangles) are shown. Values represent the mean ± S.E. from 22 cells. E, YFP-N-1/YFP-C-7 (red) exhibits partial overlap (yellow) with RhoB-labeled endosomes (green) after stimulation. Cells were transfected with s, YFP-N-1, YFP-C-7, 2AR, CFP-RhoB, and mRFP. Top row, before stimulation; bottom row, 23 min after stimulation. Bar = 10 µm. F, quantification of stimulus-dependent labeling of vesicles with YFP-N-1/YFP-C-7 and CFP-RhoB. Areas of vesicles labeled with YFP-N-1/YFP-C-7 (filled circles), CFP-RhoB (open circles), and both YFP-N-1/YFP-C-7 and CFP-RhoB (filled triangles) are shown. Values represent the mean ± S.E. from 27 cells. Videos for A, C, and E of this figure are available under Supplemental Materials. YN indicates YFP-N and YC indicates YFP-C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Production of a Functional Fluorescent G Protein _s Subunit—Because the amino and carboxyl termini of G protein subunits are important for interactions with receptors, effectors, the G protein subunits, and the plasma membrane (38-40), we attempted to produce a functional _s-GFP by inserting GFP into _s at the position corresponding to an insertion site that produced a functional _q-GFP (41), but this fusion protein did not have activity (data not shown). We then used a random insertion approach that utilized a synthetic transposon containing GFP within the Tn5 transposon. Surprisingly, the most functional fluorescent fusion protein obtained contained the GFP within the A helix of _s, whereas several insertions in exposed loops were not functional. This functional fusion protein was not expressed as well as _s was, but, when corrected for expression level, had comparable activity to that of _s (42).

In parallel with the random insertion approach, we produced an _s-CFP fusion (Fig. 1A), based on a report that inserting GFP in the 1/A loop, the site of alternative splicing in _s, leaves function intact (20). _s-CFP differs from the previously published _s-GFP construct in that it contains a Ser-Gly-Gly-Gly-Gly-Ser linker on each side of the inserted CFP. We found that such a linker was essential for producing an _q-GFP fusion that had the same expression and activity levels as those of _q (41). The expression levels (Fig. 1B) and activities (Fig. 1C) of _s and _s-CFP were similar, and _s-CFP produced a fluorescent signal in the plasma membranes of HEK-293 cells (Fig. 2). As with _q-GFP, the Ser-Gly-Gly-Gly-Gly-Ser linkers appear to be important for activity, because the activity of _s-CFP was greater than that of a comparable _s-GFP fusion lacking these linkers (20) (Fig. 1D), although the expression levels of the two fusion proteins were similar (data not shown).

Production of a Functional Fluorescent G Protein ₁₇ Complex—We applied the strategy of BiFC (22) to visualize ₁₇ dimers (23). BiFC involves the production of a fluorescent signal by two nonfluorescent fragments of YFP when they are brought together by interactions between proteins fused to each fragment. Briefly, a functional fluorescent ₁₇ dimer was produced by fusing an amino-terminal YFP fragment (residues 1-158, referred to as YFP-N) to the amino terminus of ₁ and a carboxyl-terminal YFP fragment (residues 159-238, referred to as YFP-C) to the amino terminus of ₇. When expressed together, YFP-N-₁ and YFP-C-₇ produced a fluorescent signal in the plasma membrane (Fig. 2) that was not seen when either subunit was expressed alone (23). Functionality of YFPN-/YFP-C- complexes was demonstrated by the ability to potentiate activation of adenylyl cyclase by _s in COS-7 cells (23). The BiFC method assures exclusive visualization of ₁₇ dimers rather than individual subunits and leads to more selective labeling of the plasma membrane than is obtained with individually tagged subunits, which when co-expressed, are not functional (23).

Stimulation of the ₂-AR Leads to Internalization of Both _s and ₁₇ and Substantial Co-localization of These G_s Subunits on Vesicles—To visualize and investigate the regulation of the subcellular localization patterns of the subunits of G_s during signaling, we imaged _s-CFP and YFP-N-₁/YFP-C-₇ after agonist stimulation in transfected HEK-293 cells. Stimulation of cells co-expressing _s-CFP, YFP-N-₁/YFP-C-₇, and the ₂AR with the -adrenergic agonist, isoproterenol, resulted in internalization of both _s-CFP (red in Fig. 2) and YFP-N-₁/YFP-C-₇ (green in Fig. 2) from the plasma membrane. Initially, upon leaving the plasma membrane, _s-CFP and YFPN-₁/YFP-C-₇ exhibited a diffuse staining pattern in the cytoplasm (Fig. 2, 3 min). Subsequently, the signals for these G_s subunits exhibited significant co-localization (in yellow) on intracellular vesicles (Fig. 2, 24 min).

To quantify the distribution and movements of the labeled proteins following stimulation, we developed image analysis protocols to measure the intensity, area, and overlap of labeled cellular features. The pixels in each time course image that corresponded to a specific cellular feature such as the plasma membrane or intracellular vesicles were marked or "segmented" and analyzed as described under "Experimental Procedures." The amounts of _s-CFP and YFP-N-₁/YFP-C-₇ signal that moved out of the plasma membrane upon stimulation were quantified in cells co-expressing _s-CFP or YFP-N-₁/YFP-C-₇ and a plasma membrane marker (YFP-Mem or CFP-Mem) to identify plasma membrane pixels and to control for changes in cell shape during the responses. The plasma membrane signals for _s-CFP (Fig. 3A, filled circles) and YFP-N-₁/YFP-C-₇ (Fig. 3A, filled squares) decreased with the same kinetics and the responses were essentially complete by 6 min. In the absence of stimulation, changes in the plasma membrane intensity of _s-CFP (Fig. 3A, open circles) and YFP-N-₁/YFP-C-₇ (Fig. 3A, open squares) did not occur.

The amounts of _s-CFP and YFP-N-₁/YFP-C-₇ signal that stained the cytoplasm diffusely were quantified relative to that of a cytoplasm marker (mRFP) and were determined for intracellular pixels, excluding vesicles that became labeled and the nucleus (see "Experimental Procedures"). This analysis demonstrated that the cytoplasm signals for both _s-CFP (Fig. 3B, circles) and YFP-N-₁/YFP-C-₇ (Fig. 3B, squares) increased upon stimulation at rates similar to that at which they decreased in the plasma membrane. Labeling of vesicles with _s-CFP and YFP-N-₁/YFP-C-₇ was quantified by determining the per cell areas of the labeled vesicles and the extent to which vesicles labeled with the two signals overlapped (Fig. 3C). The total areas of vesicles labeled with either _s-CFP (Fig. 3C, circles) or YFP-N-₁/YFP-C-₇ (Fig. 3C, squares) increased with the same kinetics and to the same extent. Approximately 50% of the vesicle areas contained both signals (Fig. 3C, triangles). This is an underestimate of the overlap of _s-CFP and YFP-N-₁/YFP-C-₇ on vesicles, because vesicle movement in between acquisition of the sequential images reduced the measured overlap.

Stimulus-dependent decreases in the plasma membrane intensity of _s-CFP were somewhat greater in magnitude than those of YFP-N-₁/YFP-C-₇ (Fig. 3A) and the increases in cytoplasm signal were greater for _s-CFP than for YFP-N-₁/YFP-C-₇ (Fig. 3B). However, because the stimulus-dependent changes in the intensities of the two signals in these locations were determined relative to each of their starting values and do not represent absolute amounts of the proteins, these changes in the intensities of _s-CFP and YFP-N-₁/YFP-C-₇ are not directly comparable. Nevertheless, the similar timing of the _s-CFP and YFP-N-₁/YFP-C-₇ internalization responses (Fig. 3) and the similar localization patterns of these G protein subunits in the diffuse cytoplasm and on vesicles (Fig. 2) suggest that the activated populations may traffic together.

Vesicles Labeled with _s and ₁₇ Exhibit Minimal Overlap with ₂AR-labeled Vesicles—The trafficking patterns of the G_s subunits were compared directly with that of the ₂AR in cells that co-expressed the labeled proteins. In cells co-expressing _s-CFP, unlabeled ₁₇, and ₂AR-GFP, there was minimal overlap between vesicles labeled with _s-CFP (red in Fig. 4A) and those labeled with ₂AR-GFP (green in Fig. 4A). Image analysis of these cells demonstrated that similar amounts of vesicles, as defined by their per cell areas, became labeled with _s-CFP (Fig. 4B, filled circles) and ₂AR-GFP (Fig. 4B, open circles), despite the minimal overlap of the two vesicle populations (Fig. 4B, filled triangles). Similarly, in cells that co-expressed unlabeled _s, YFP-N-₁/YFP-C-₇, and ₂AR-CFP, there was minimal overlap between vesicles labeled with YFPN-₁/YFP-C-₇ (red in Fig. 4C) and those labeled with ₂ARCFP (green in Fig. 4C). Again, image analysis showed that similar amounts of vesicles became labeled with YFP-N-₁/YFP-C-₇ (Fig. 4D, filled circles) and ₂AR-CFP (Fig. 4D, open circles), although the two vesicles populations did not co-localize (Fig. 4D, filled triangles). In addition, the fluorescent G_s subunits (filled circles in Fig. 4, B and D) labeled vesicles more rapidly than the fluorescent ₂AR (open circles in Fig. 4, B and D).

View larger version (69K): [in this window] [in a new window]   FIG. 4. Vesicles labeled with s-CFP and YFP-N-1/YFP-C-7 do not co-localize well with 2AR-GFP- and 2AR-CFP-labeled vesicles, respectively. A, s-CFP (red) exhibits minimal overlap (yellow) with 2AR-GFP (green) after stimulation. Cells were transfected with s-CFP, 1, 7, and 2AR-GFP. Top row, before stimulation; bottom row, 28 min after stimulation. Bar = 10 µm. B, quantification of stimulus-dependent labeling of vesicles with s-CFP and 2AR-GFP. Areas of vesicles labeled with s-CFP (filled circles), 2AR-GFP (open circles), and both s-CFP and 2AR-GFP (filled triangles) are shown. Values represent mean ± S.E. from 20 cells. C, YFP-N-1/YFP-C-7 (red) exhibits minimal overlap (yellow) with 2AR-CFP (green) after stimulation. Cells were transfected with s, YFP-N-1, YFP-C-7, and 2AR-CFP. Top row, before stimulation; bottom row, 15 min after stimulation. Bar = 10 µm. D, quantification of stimulus-dependent labeling of vesicles with YFP-N-1/YFP-C-7 and 2AR-CFP. Areas of vesicles labeled with YFP-N-1/YFP-C-7 (filled circles), 2AR-CFP (open circles), and both YFP-N-1/YFP-C-7 and 2AR-CFP (filled triangles) are shown. Values represent mean ± S.E. from 20 cells. Videos for A and C of this figure are available as Supplemental Material.

Receptor-stimulated Internalization of the ₂AR, but Not _s or , Is Inhibited by a Dominant Negative Dynamin 1 Mutant

Vesicles Labeled with the ₂AR Exhibit Greater Co-localization with RhoB-labeled Endosomes Than Do Vesicles Labeled with _s or ₁₇—The initial diffuse labeling of the cytosol by both _s-CFP and YFP-N-₁/YFP-C-₇ after stimulation and the inability of Dyn1(K44A)-mRFP to block their internalization from the plasma membrane suggested that these G_s subunits may not directly internalize on vesicles. However, the subsequent labeling of vesicles by both _s-CFP and YFP-N-₁/YFP-C-₇ suggested that vesicular trafficking may play a role in effector modulation and/or the recycling of G_s back to the plasma membrane. Although G_s subunit-labeled vesicles did not co-localize well with ₂AR-labeled vesicles (Fig. 4), we investigated the possibility that the G_s subunit-labeled vesicles might be endosomes, because diverse internalization pathways can converge on endosomes (45).

To explore the nature of the vesicles labeled by the G_s subunits, stimulus-dependent overlap of the G_s subunits and the ₂AR with RhoB-labeled endosomes was measured. RhoB has been reported to associate with early endosomes (34) and multivesicular bodies, a prelysosomal compartment thought to be involved in sorting internalized receptors for degradation (35). Vesicles labeled with ₂AR-GFP that formed after stimulation occupied similar per cell areas as did CFP-RhoB-labeled endosomes, which exhibited unchanged areas upon stimulation (Fig. 6, A and B). After stimulation, 50% of the RhoB-labeled vesicle area overlapped with that of ₂AR-labeled vesicles (Fig. 6, A and B). As with _s-CFP and YFP-N-₁/YFP-C-₇ vesicles (Figs. 2 and 3), the actual overlap was underestimated, because of the sequential acquisition of images. In contrast, although vesicles labeled with _s-CFP or YFP-N-₁/YFP-C-₇ occupied similar per cell areas, respectively, as did YFP-RhoB- and CFP-RhoB-labeled endosomes, the G_s subunit vesicles co-localized with only 25% of the RhoB-labeled endosome area (Fig. 6, C-F).

Vesicles Labeled with _s and ₁₇ Exhibit Greater Co-localization with Rab11-labeled Endosomes Than Do Vesicles Labeled with the ₂AR—Some of the internalized _s-CFP and YFP-N-₁/YFP-C-₇ localized to the Golgi apparatus region, but did not overlap directly with the trans-medial Golgi region, labeled with YFP or CFP, respectively, fused to the amino-terminal 81 residues of 1,4-galactosyltransferase (46, 47) (not shown). Similar results were obtained using a GFP-tagged marker for the cis-Golgi region, GFP-GM130 (48, 49) (not shown). To further investigate the significance of the appearance of _s-CFP and YFP-N-₁/YFP-C-₇ in this cellular region, we tested for overlap with Rab11, which localizes to recycling endosomes and post-Golgi membranes, including the trans-Golgi network, and secretory vesicles (24, 50), and plays a role in regulating recycling through the slow perinuclear recycling endosome route (24).

CFP- and YFP-Rab11 (31) selectively labeled a small perinuclear region (Fig. 7, A, C, and E). Segmentation analysis demonstrated that the cellular area occupied by Rab11-labeled vesicles (open circles in Fig. 7) was much smaller than that occupied by Rho-B-labeled vesicles (open circles in Fig. 6). However, we observed significant stimulus-dependent overlap in the perinuclear region between _s-CFP and YFP-Rab11 (Fig. 7, A and B). Similar overlap was obtained with YFP-N-₁/YFP-C-₇ and CFP-Rab11 (Fig. 7, C and D). Approximately 40% of the Rab11-labeled vesicles co-localized with G_s-subunit-labeled vesicles by the ends of the time courses. The total area of overlap between Rab11-labeled vesicles and those labeled with G_s subunits (filled triangles in Fig. 7, B and D) was less than that between RhoB- and G_s subunit-labeled vesicles (filled triangles in Fig. 6, D and F). However, a greater percentage of the Rab11-labeled vesicles than the RhoB-labeled vesicles co-localized with G_s subunit-labeled vesicles. In contrast, there was minimal overlap between ₂AR-GFP and CFP-Rab11 (Fig. 7, E and F), confirming previous studies showing that the ₂AR recycles to the plasma membrane directly from early endosomes via a Rab4-dependent mechanism (51), rather than through the slow perinuclear recycling endosome route.

View larger version (62K): [in this window] [in a new window]   FIG. 7. Vesicles labeled with s-CFP and YFP-N-1/YFP-C-7, but not 2AR-GFP exhibit partial co-localization with Rab11-labeled endosomes. A, s-CFP (red) exhibits overlap (yellow) in the perinuclear region with YFP-Rab11 (green) after stimulation. Cells were transfected with s-CFP, 1, 7, 2AR, YFP-Rab11, and mRFP. Top row, before stimulation; bottom row, 24 min after stimulation. Cell border is blue. Bar = 10 µm. B, quantification of stimulus-dependent labeling of vesicles with s-CFP and YFP-Rab11. Areas of vesicles labeled with s-CFP (filled circles), YFP-Rab11 (open circles), and both s-CFP and YFP-Rab11 (filled triangles) are shown. Values represent the mean ± S.E. from 20 cells. C, YFP-N-1/YFP-C-7 (red) exhibits overlap (yellow) in the perinuclear region with CFP-Rab11 (green) after stimulation. Cells were transfected with s, YFP-N-1, YFP-C-7, 2AR, CFP-Rab11, and mRFP. Top row, before stimulation; bottom row, 28 min after stimulation. Bar = 10 µm. D, quantification of stimulus-dependent labeling of vesicles with YFP-N-1/YFP-C-7 and CFP-Rab11. Areas of vesicles labeled with YFP-N-1/YFP-C-7 (filled circles), CFP-Rab11 (open circles), and both YFP-N-1/YFP-C-7 and CFP-Rab11 (filled triangles) are shown. Values represent the mean ± S.E. from 20 cells. E, 2AR-GFP (red) does not overlap with CFP-Rab11 (green) after stimulation. Cells were transfected with s, 1, 7, 2AR-GFP, CFP-Rab11, and mRFP. Top row, before stimulation; bottom row, 26 min after stimulation. Bar = 10 µm. F, quantification of stimulus-dependent labeling of vesicles with 2AR-GFP and CFP-Rab11. Areas of vesicles labeled with 2AR-GFP (filled circles), CFP-Rab11 (open circles), and both 2AR-GFP and CFP-Rab11 (filled triangles) are shown. Values represent mean ± S.E. of 20 cells. Videos for A, C, and E of this figure are available under Supplemental Materials. YN indicates YFP-N and YC indicates YFP-C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A previous study using immunohistochemistry demonstrated activation-dependent redistribution of _s to the cytosol in HEK-293 cells stably transfected with the ₂AR and HA-tagged _s (19). The staining pattern for internalized _s was punctate, but could be distinguished from that of the ₂AR. In a study using a different _s-GFP construct, isoproterenol stimulation of COS-1 cells resulted in a partial decrease in association of _s-GFP with some regions of the plasma membrane as well as insertion of _s-GFP at other membrane sites (20). Differences between our results with _s-CFP expressed in HEK-293 cells and these previous results with _s-GFP could be because of the greater activity of the _s-CFP construct and/or the different cell types examined. Stimulus-dependent internalization has not been imaged previously, but using an enzyme-linked immunosorbent assay, redistribution of from the plasma membrane to low density microsomes was observed in response to isoproterenol stimulation (21).

Our results indicate that G_s and the ₂AR dissociate upon hormonal stimulation and utilize different mechanisms for internalization from the plasma membrane, because internalization of the ₂AR, but not _s or ₁₇, can be blocked by a dominant negative dynamin 1 mutant. The inability of Dyn1(K44A)-mRFP to block internalization of _s extends a previous study in which hypertonic sucrose, which inhibits the formation of clathrin-coated pits, blocked internalization of the ₂AR, but not _s (19). Because dynamin is involved in both clathrin-mediated endocytosis (52, 53) and endocytosis of caveolae (54), our results demonstrate that internalization of _s and