Agonist-induced translocation of Gq/11alpha immunoreactivity directly from plasma membrane in MDCK cells

John M. Arthur1, Georgiann P. Collinsworth2, Thomas W. Gettys2, and John R. Raymond2

1 University of Louisville, Louisville, Kentucky 40202; and 2 Medical University of South Carolina, Charleston, South Carolina 29425


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Both Gsalpha and Gqalpha are palmitoylated and both can move from a crude membrane fraction to a soluble fraction in response to stimulation with agonists. This response may be mediated through depalmitoylation. Previous studies have not demonstrated that endogenous guanine nucleotide-binding regulatory protein (G protein) alpha -subunits are released directly from the plasma membrane. We have examined the effect of agonist stimulation on the location of Gq/11alpha immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 µM) caused Gq/11alpha , but not Gialpha , to rapidly translocate from purified plasma membranes to the supernatant. AlF and GTP also caused translocation of Gq/11alpha immunoreactivity from purified plasma membranes. BK caused translocation of Gq/11alpha immunoreactivity in intact cells from the basal and lateral plasma membranes to an intracellular compartment as assessed by confocal microscopy. Thus Gq/11alpha is released directly from the plasma membrane to an intracellular location in response to activation by an agonist and direct activation of G proteins. G protein translocation may be a mechanism for desensitization or for signaling specificity.

G protein; bradykinin; palmitoylation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HETEROTRIMERIC guanine nucleotide-binding regulatory proteins (G proteins) couple cell surface receptors to intracellular second messengers. The alpha -subunit is covalently bound to a myristoyl (2, 9) and/or a palmitoyl (12) side chain. These fatty acyl side chains aid in membrane association and signaling (9, 34). Whereas some G proteins are both myristoylated and palmitoylated, Gq/11alpha and Gsalpha are only palmitoylated (35). As early as 1985, Rodbell proposed that G protein alpha -subunits might diffuse from the plasma membrane after activation to function as "programmable messengers" (26). That Gsalpha can translocate from a membrane fraction to the soluble phase after activation has been well documented, although no evidence of translocation of endogenously expressed G proteins directly from the plasma membrane, where the G protein would be likely to encounter an agonist-liganded receptor, has yet been provided. Two lines of evidence suggest that G proteins can move from one compartment of the cell to another. First, Gsalpha can translocate from crude cell particulate fractions to cytoplasm after agonist stimulation. Activation of Gsalpha by several agents has been shown to increase the cytosolic content of Gsalpha in S49 and mouse mastocytoma cells (11, 21, 23) and decrease the Gsalpha immunoreactivity in crude membrane fractions by up to 75% (16, 22). Although those studies show an agonist-induced dissociation of Gsalpha from a cellular particulate fraction, a definite identification of the plasma membrane as the source of the G proteins has not yet been established. Recently, translocation of transfected HA-tagged Gsalpha from the plasma membrane in response to beta -adrenergic receptor stimulation has been shown by immunofluorescence microscopy (33).

Similarly, members of the Gq/11alpha family have also been shown to translocate in intact cells. Levels of Gqalpha immunoreactivity in crude membrane fractions have been shown to decrease after 16-h treatment with carbachol in CHO cells (18) and gonadotropin-releasing hormone (GnRH) in pituitary cells (29). In HEK cells transfected with G11alpha subunits and thyrotropin-releasing hormone (TRH) receptors, stimulation with TRH caused a time-dependent increase in G11alpha immunoreactivity in the cytosolic fraction and low-density membranes and a decrease in G11alpha immunoreactivity in plasma membrane fractions (30). Total G11alpha immunoreactivity decreased during this time. In CHO cells transfected with muscarinic receptors, stimulation with carbachol for 30 min resulted in a decrease in Gqalpha and G11alpha immunoreactivity in a plasma membrane fraction and an increase in the light vesicular membrane fraction (31). A second line of evidence supporting the functional importance of membrane attachment of G proteins derives from studies of their lipid anchors. Lipid anchors have been shown to be important for targeting and maintenance of Gsalpha and Gqalpha in cellular particulate fractions (13, 34). Stimulation of beta 2-adrenergic receptors in COS and S49 cells induces a dynamic cycle of palmitoylation and depalmitoylation of Gsalpha (5, 19, 32). The importance of membrane attachment is underscored by a study in which cysteine residues near the amino-terminal end of constitutively active Gsalpha or Gqalpha were replaced. These mutations prevented the G proteins from being palmitoylated or membrane bound. Loss of membrane binding abolished the ability of Gqalpha to activate phospholipase C and reduced formation of cAMP by Gsalpha (34). This study was designed to determine whether stimulation of endogenous receptors and G proteins causes translocation in the Madin-Darby canine kidney (MDCK) cell model of renal distal tubule. We demonstrate that bradykinin (BK) causes translocation of G protein alpha -subunits of the Gq class from the plasma membrane in whole cells and in purified plasma membrane.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of plasma membranes. Purified plasma membranes were prepared by aqueous two-phase partition through a polyethylene glycol-dextran gradient as previously described (17, 20). The yield of plasma membranes was 1-8% relative to the initial quantity of MDCK cell crude membranes. To determine the degree of enrichment of plasma membranes, we measured K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase activity as previously described (10, 20). Two-phase partition resulted in an enrichment of 130-fold in plasma membrane marker enzyme activity.

Western blot. The purified plasma membranes were stimulated with BK (0.1 µM), AlF (0.75 mM NaF and 5 µM AlCl3), or GTP (10 µM) for 10 min at 37°C in a buffer containing 30 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl, 3 µM GDP, 1 mM benzamidine, and 5 µg/ml soybean trypsin inhibitor. The plasma membrane and supernatant fractions were separated by centrifugation at 16,000 g for 15 min. In two studies, an ultracentrifuge was used to obtain a 100,000 g pellet and supernatant fractions. For these studies the pellet was resuspended in a volume equal to that of the pellet (50 µl). The supernatant was subjected to SDS-PAGE and Western blot as previously described (1) using an antiserum directed against the carboxyl-terminal region shared by Gqalpha and G11alpha (QLNLKEYNLV). This antibody is specific for Gq/11alpha and does not crossreact with Galpha i-1, Galpha i-2, or Galpha i-3 (data not shown). In additional studies, Galpha i-1,2- and Galpha i-3-specific antisera were used to determine whether these G proteins translocated in response to agonists. These antibodies have been previously characterized (24). Peroxidase-conjugated goat anti-rabbit IgG (Sigma) was used as a secondary antibody (1:10,000) and the ECL chemiluminescent system (Amersham, Arlington Heights, IL) was used to visualize G protein immunoreactivity. Film was exposed for 5-30 min to obtain densities in the linear range of the film. Densitometric intensity of the control and stimulated bands was determined using a model GS-670 imaging densitometer (Bio-Rad, Hercules, CA).

Immunofluorescence. MDCK cells plated on glass coverslips and grown to 75% confluence in MEM media containing 10% fetal calf serum were pretreated for 1 h with serum-free media containing 0.5% BSA. Cells were stimulated with BK for 10 min in a buffer containing 30 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl, 1 mM benzamidine, and 5 µg/ml soybean trypsin inhibitor. The cells were then washed twice with cold PBS, permeabilized with a buffer containing 0.05% saponin, fixed with 3% paraformaldehyde in PBS, quenched with 50 mM NH4Cl in PBS, and incubated with PBS/10% FCS as described (7). Cells were then incubated for 1 h with the Gq/11alpha antibody (1:50, PBS/5% FCS), washed six times, and then incubated for 1 h with FITC-conjugated goat anti-rabbit IgG (Sigma). Cells were rinsed and mounted on glass slides with vectashield (Vector Labs, Burlingame, CA). We visualized Gq/11alpha immunoreactivity using a Zeis laser confocal microscope with emission at 515 nm. A Z series was done with an image obtained every 1 µm. Specificity of the antibody was determined by controls utilizing preimmune serum, a monoclonal antibody against gamma -catenin (a component of tight junctions; Transduction Laboratories, Lexington, KY), the Gq/11alpha immunogen peptide, and an irrelevant peptide (IEKFREEAEERDICIC).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies were designed to test the hypothesis that G proteins of the Gqalpha family are released from the plasma membrane in response to stimulation with BK. To establish that activated G proteins are released directly from the plasma membrane, rather than from some other cellular particulate fraction such as endoplasmic reticulum or Golgi, we stimulated highly enriched plasma membrane fractions derived from MDCK cells with BK. After centrifugation, we performed a Western blot analysis on the supernatant fraction to determine whether G proteins had been released from the membrane. Exposure of plasma membranes to BK for 10 min resulted in an increased release of Gq/11alpha immunoreactivity into the supernatant (Fig. 1A), from 1.25 ± 0.56 to 2.82 ± 0.81 densitometric units (n = 9, P < 0.05). BK caused an average percent increase in Gq/11alpha immunoreactivity in the supernatant of 170 ± 53%. This translocation of Gq/11alpha immunoreactivity represents a movement of G proteins out of the plasma membranes. In additional studies, the ability of BK to induce the translocation of other G proteins was examined. Western blots were done on the supernatant fraction with antibodies that recognize Galpha i-1,2 and Galpha i-3. Unlike the results seen with Gq/11alpha , no Galpha i-1,2 or Galpha i-3 immunoreactivity was detected in the supernatant fraction after a 10-min treatment with buffer alone. In addition, BK did not cause the release of Galpha i-1,2 or Galpha i-3 immunoreactivity from the plasma membranes (data not shown).


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Fig. 1.   A: bradykinin (BK) stimulation increased release of Gq/11alpha immunoreactivity from plasma membranes. Madin-Darby canine kidney (MDCK) cell plasma membranes were treated for 10 min with vehicle (-) or 0.1 µM BK (+). Amount of Gq/11alpha released from plasma membrane was determined by Western blot of 16,000 g soluble fraction. B: stimulation of BK receptor, as well as direct stimulation of guanine nucleotide-binding regulatory proteins (G proteins) by GTP and AlF, caused translocation of Gq/11alpha immunoreactivity out of plasma membrane at 10 min. Amount of immunoreactivity appearing in 16,000 g soluble fraction after treatment of plasma membranes with vehicle or indicated drug was compared for BK (n = 9), GTP (n = 3), and AlF (n = 4).

To determine whether translocation of Gq/11alpha immunoreactivity could be caused by direct activators of G proteins, we stimulated plasma membranes with GTP or AlF (Fig. 1B). GTP increased Gq/11alpha immunoreactivity in the supernatant fraction from 1.32 ± 0.22 to 3.16 ± 0.44 U (n = 3, P < 0.05), representing an increase of 153 ± 64% in translocation. AlF increased Gq/11alpha immunoreactivity in the supernatant fraction from 1.34 ± 0.15 to 2.27 ± 0.25 U (n = 4, P < 0.05), representing an increase of 78 ± 36% in Gq/11alpha immunoreactivity in the soluble fraction. These data indicate that both receptor- and nonreceptor-mediated G protein activation cause accelerated translocation of Gq/11alpha immunoreactivity out of the plasma membrane.

To ensure that the Gq/11alpha immunoreactivity remaining in the supernatant was not the result of incomplete centrifugation, we repeated these studies using an ultracentrifuge. BK stimulation resulted in an increase in release of Gq/11alpha immunoreactivity into the 100,000 g supernatant (Fig. 2). The fraction of Gq/11alpha immunoreactivity appearing in the supernatant was compared with that remaining in the pellet after centrifugation at 100,000 g for 20 min. To more accurately determine the relative amounts of immunoreactivity in the pellet and supernatant, we ran lanes containing equivalent amounts of supernatant and pellet as well as lanes that contained a 1:10 dilution of the pellet. Incubation with buffer alone resulted in translocation of 2% of Gq/11alpha immunoreactivity after 10 min, whereas incubation with BK resulted in 16% translocation of Gq/11alpha immunoreactivity.


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Fig. 2.   Stimulation with BK caused an increase in release of Gq/11alpha immunoreactivity from plasma membrane and a reciprocal decrease in immunoreactivity remaining in pellet. Western blot (A) and densitometric analysis (B) are shown. In these studies, plasma membranes were stimulated with 0.1 µM BK as described, then centrifuged at 100,000 g. Lanes labeled supernatant and pellet 1:1 contained equivalent fractions of soluble and particulate fractions, respectively, whereas lanes labeled pellet 1:10 contained only 10%. Representative of 2 experiments.

We assessed the rapidity with which this response occurred by measuring the amount of immunoreactivity appearing in the soluble fraction with an increasing duration of stimulation (Fig. 3). There was a gradual increase in the amount of translocation induced by BK over the first 10 min. The response was maximal by 10 min and did not increase further up to 30 min. Thus BK induces a time-dependent increase in translocation of Gq/11alpha immunoreactivity out of the plasma membrane. These studies establish the possibility that Gq/11alpha activation leads to its release directly from the plasma membrane. Because these studies were done in an artificial system that lacks cytoskeletal and other membrane support components, it is unclear whether similar translocations occur directly from the plasma membrane in intact cells.


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Fig. 3.   Translocation of Gq/11alpha immunoreactivity in response to stimulation with BK is time dependent. Amount of immunoreactivity appearing in 16,000 g soluble fraction was determined after indicated period of stimulation with BK (0.1 µM).

To determine whether translocation of Gq/11alpha immunoreactivity occurred in intact cells, as well as from purified plasma membranes, we used immunofluorescence confocal microscopy to study the intracellular location of Gq/11alpha before and after agonist treatment. We confirmed specificity of the antibody by the failure of the preimmune serum to label G proteins and the ability of the immunogenic peptide, but not an irrelevant peptide, to block immunofluorescence (Fig. 4). We next examined the ability of BK to cause translocation of Gq/11alpha immunoreactivity away from the membrane. Cross-sectional images were obtained at 1-µm intervals from the basal to the apical surface in control and BK-stimulated MDCK cells. Before treatment with BK, Gq/11alpha immunoreactivity was located along the basal surface of the cell near its interface with the coverslip. A large amount of immunoreactivity could also be seen extending up the plasma membrane at the lateral surfaces of the cell. A small amount of immunoreactivity was seen inside the cells (Fig. 5, A-D). This finding was consistent with G protein localization at the cell surface, where it could interact with both receptors and effectors. After BK treatment, Gq/11alpha immunoreactivity could still be seen at the basal and lateral surfaces, but much of the immunoreactivity had shifted to a predominantly intracellular location (Fig. 5, E-H). Punctate localization of Gq/11alpha immunoreactivity could be seen throughout the cytoplasm. There was a focal region of intracellular clearing that may represent exclusion of Gq/11alpha from the nucleus. These studies demonstrate, using two separate techniques, that BK stimulation causes Gq/11alpha to move rapidly from the plasma membrane to an intracellular compartment or compartments.


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Fig. 4.   Confocal immunofluorescence micrograph of Gq/11alpha and gamma -catenin immunoreactivity. Gq/11alpha antibody recognized a protein localized primarily to lateral border of cell. Cells were treated as follows. A: 1° antibody-preimmune rabbit serum (1:100), 2° antibody-Oregon green-conjugated anti-rabbit (1:200). B: 1° antibody-polyclonal anti-Gq/11alpha (1:100), 2° antibody-Oregon green-conjugated anti-rabbit (1:200). C: 1° antibody-polyclonal anti-Gq/11alpha (1:100) and monoclonal anti-gamma -catenin, 2° antibody-Oregon green-conjugated anti-rabbit (1:200), and tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated anti-mouse (1:50). D: 1° antibody-polyclonal anti-Gq/11alpha (1:100), monoclonal anti-gamma -catenin, and Gq/11alpha immunizing peptide (1 mg/ml), 2° antibody-Oregon green-conjugated anti-rabbit (1:200), and TRITC-conjugated anti-mouse (1:50). E: 1° antibody-polyclonal anti-Gq/11alpha (1:100), monoclonal anti-gamma -catenin, and Gq/11alpha -irrelevant peptide (IEKFREEAEERDICIC; 1 mg/ml), 2° antibody-Oregon green-conjugated anti-rabbit (1:200), and TRITC-conjugated anti-mouse (1:50).


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Fig. 5.   Confocal immunofluorescence micrographs of MDCK cells treated for 10 min with vehicle (A-D) or 0.1 µM BK (E-H). Gq/11alpha immunoreactivity can be seen at basal surface in both control (A) and stimulated (E) cells. B-D and F-H represent sequential 2-µm cuts moving apically from basal surface. In control cells, most Gq/11alpha immunoreactivity is around lateral edges of cell. After stimulation with BK, a much larger share of Gq/11alpha immunoreactivity is seen within cell. Photograph is typical of results seen in 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first direct demonstration of movement of endogenous G proteins from the plasma membrane to another intracellular compartment. Previous studies have shown either movement out of a crude membrane fraction or increased appearance in whole cell low-density fractions associated with decreased immunoreactivity in plasma membrane fractions. The subcellular site of origin of these G proteins has not previously been described. G proteins could be released from other organelles present in crude membrane fractions, such as Golgi or endoplasmic reticulum. We used both a purified fraction of plasma membranes and direct visualization with a confocal microscope to determine whether plasma membrane was the source of G protein translocation. Although purified plasma membranes do contain contaminating amounts of other membranes including Golgi, endoplasmic reticulum, and mitochondria (17), the membranes used in the current study showed a 130-fold increase in plasma membrane marker enzyme activity. Therefore, it is unlikely that the magnitude of G protein release demonstrated in this study could have come from contaminating membranes.

In the present study Gq/11alpha moved from the plasma membrane to the supernatant in response to stimulation with BK. We have demonstrated that this translocation occurs from plasma membranes, using two techniques. Translocation occurs from a purified preparation of plasma membranes, which lack cytoskeletal support elements, and from the plasma membrane in whole cells, as seen with confocal microscopy. Peak translocation by BK from plasma membranes occurs by 10 min. In contrast to the translocation of Gq/11alpha , BK did not cause detectable translocation of Gialpha subunits, although it is possible that a very small amount of translocation occurred that was below the limit of detection.

In these studies, no additional GTP was added to the plasma membrane preparations. Although G protein activation requires GTP binding to the alpha -subunit, translocation occurred without additional GTP in the buffer. Thus either sufficient quantities of GTP must be present in the preparation or translocation of G proteins can occur without GTP. Concentrations of GTP have not been measured in purified plasma membranes and may vary significantly in the vicinity of proteins with nucleotide binding abilities. Nucleotide content has been measured associated with the F1-ATPase enzyme purified from the bacteria Micrococcus lysodeikticus, in which GTP content was found to be 8.6 µM (14). These data indicate that significant concentrations of GTP may be present in the microenvironment of the G protein, even though the GTP concentration of the total purified plasma membrane is miniscule. GTP content has also been measured in rod outer segments prepared to have leaky membranes. In this preparation, which is rich in G proteins, GTP was also present in significant amounts (27). The concentration of GTP at the G protein in purified plasma membranes of MDCK cells is not known, but based on these studies, it may be significant. Thus BK and endogenous GTP may work synergistically by interaction of an agonist-liganded BK receptor with a G protein, followed by the binding of GTP to the G protein, to cause translocation. These results demonstrate that either agonist binding of the receptor or direct G protein activation causes translocation of G proteins. Agonist binding of the receptor is not required for translocation because the addition of AlF, which mimics the GTP-bound state of the G protein, causes translocation.

The mechanism of translocation is unknown; however, cleavage of the thioester bond and release of the palmitate moiety is one potential mechanism. Palmitoylation of G proteins is required for localization to the plasma membranes (13, 34). Palmitoylation and depalmitoylation of G proteins is a dynamic process that is stimulated by agonists (15). A palmitoyl protein thioesterase that cleaves palmitate has been described (3, 4). Activation of the BK receptor could cause cleavage of the palmitoyl-thioester linkage and release of the plasma membrane-anchoring palmitate by one of two possible mechanisms. Receptor activation could cause activation of the thioesterase or it could lead to a conformational change in the G protein that would make the thioester bond more accessible to the thioesterase. Which of these mechanisms might be responsible for cleavage of the anchoring palmitate is not known. It is interesting that activation by BK, which should only affect a subset of G proteins, produced a similar magnitude of translocation to that produced by AlF, which could potentially activate all G proteins. This suggests that G protein activation alone is not as potent a stimulus to translocation as is receptor-mediated activation. The role of depalmitoylation in translocation of Gq/11alpha in response to stimulation with BK has not yet been determined, but it seems a likely mechanism in light of the known role of palmitoyl residues in membrane targeting of Gq/11alpha and Gsalpha .

The physiological role of translocation of G proteins away from the plasma membrane is unclear. Translocation could be a means of desensitization, degradation, or recycling of G proteins, or could be important in mediating physiological responses. A decrease in the levels of Gq/11alpha in the plasma membrane might attenuate the responses mediated through those proteins. In support of this hypothesis, long-term stimulation of GnRH or muscarinic receptors causes a decrease in Gq/11alpha immunoreactivity in alpha T3-1 or CHO cell extracts (18, 28, 29). In addition, translocated G proteins could be physically associated with the receptor and/or effectors such as phospholipase C. Movement of these proteins out of the membrane would further attenuate the response to stimulation. Alternatively, Gqalpha could be involved in a signal transduction pathway that requires it to dissociate from the membrane. No direct role requiring dissociated G protein alpha -subunits has been described, although it has been proposed that Gsalpha may dissociate from the intestinal brush-border membrane in response to ADP-ribosylation by cholera toxin and traverse to the basolateral surface where it could activate adenylyl cyclase (6). Recently translocation of transfected Gsalpha from the plasma membrane in response to beta -adrenergic receptor stimulation has been shown by immunofluorescence microscopy. This translocation is reversed by treatment with receptor antagonists (33). This reversibility could be consistent with a physiological role for G protein translocation in response to agonist stimulation. Because MDCK cells are a model for renal distal tubule (25), basal-to-apical translocation could allow receptor stimulation at one surface of the renal tubule to have specific effects at other cellular sites. G protein alpha -subunits are differentially expressed on different surfaces of MDCK cells (8). The current studies document that activation of Gq/11alpha results in their direct release from plasma membranes, suggesting important mechanistic implications for the function of these G proteins. Elucidation of the function of G protein translocation will require further studies but could serve as a mechanism for desensitization or to produce a specific effect at other locations within the cell.


    ACKNOWLEDGEMENTS

J. R. Raymond was supported by NIH Grant NS-29037, a Veterans Affairs Merit Award, and a Grant-in-Aid from the American Heart Association. T. W. Gettys was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42486 and a Research Grant from the American Diabetes Association. J. M. Arthur was the recipient of a National Kidney Foundation/American Society of Nephrology/Sandoz research fellowship.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. M. Arthur, Kidney Disease Program, 615 S. Preston St., Louisville, KY 40202 (E-mail: jarthur{at}kdp01.kdp-baptist.louisville.edu).

Received 7 May 1998; accepted in final form 10 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arthur, J. M., G. P. Collinsworth, T. W. Gettys, L. D. Quarles, and J. R. Raymond. Specific coupling of a cation-sensing receptor to G protein alpha -subunits in MDCK cells. Am. J. Physiol. 273 (Renal Physiol. 42): F129-F135, 1997[Abstract/Free Full Text].

2.   Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. Myristoylated alpha  subunits of guanine nucleotide-binding regulatory proteins. Proc. Natl. Acad. Sci. USA 84: 7493-7497, 1987[Abstract].

3.   Camp, L. A., and S. L. Hofmann. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-ras. J. Biol. Chem. 268: 22566-22574, 1993[Abstract/Free Full Text].

4.   Camp, L. A., L. A. Verkruyse, S. J. Afendis, C. A. Slaughter, and S. L. Hofmann. Molecular cloning and expression of palmitoyl-protein thiosterase. J. Biol. Chem. 269: 23212-23219, 1994[Abstract/Free Full Text].

5.   Degtyarev, M. Y., A. M. Spiegel, and T. L. Z. Jones. Increased palmitoylation of the Gs protein alpha  subunit after activation by the beta -adrenergic receptor or cholera toxin. J. Biol. Chem. 268: 23769-23772, 1993[Abstract/Free Full Text].

6.   Dominguez, P., G. Velasco, F. Barros, and P. S. Lazo. Intestinal brush border membranes contain regulatory subunits of adenylyl cyclase. Proc. Natl. Acad. Sci. USA 84: 6965-6969, 1987[Abstract].

7.   Dupree, P., R. G. Parton, G. Raposo, T. V. Kurzchalia, and K. Simons. Caveolae and sorting in the trans-Golgi network of epithelial cells. EMBO J. 12: 1597-1605, 1993[Abstract].

8.   Hamilton, S. E., and N. M. Nathanson. Differential localization of G-proteins, Galpha o and Galpha i-1, -2, and -3, in polarized epithelial MDCK cells. Biochem. Biophys. Res. Commun. 234: 1-7, 1997[Medline].

9.   Jones, T. L. Z., W. F. Simonds, J. J. Merendino, M. R. Brann, and A. M. Spiegel. Myristoylation of an inhibitory GTP-binding protein alpha  subunit is essential for its membrane attachment. Proc. Natl. Acad. Sci. USA 87: 568-572, 1990[Abstract].

10.   Kashiwamata, S., S. Goto, R. K. Semba, and F. N. Suzuki. Inhibition by bilirubin of (Na+ and K+)-activated adenosine triphosphatase and K+-activated p-nitrophenylphosphatase activities of NaI-treated microsomes from young rat cerebrum. J. Biol. Chem. 254: 4577-4584, 1979[Medline].

11.   Levis, M. J., and H. R. Bourne. Activation of the alpha  subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity. J. Cell Biol. 119: 1297-1307, 1992[Abstract].

12.   Linder, M. E., P. Middleton, J. R. Hepler, R. Taussig, A. G. Gilman, and S. M. Mumby. Lipid modifications of G proteins: alpha  subunits are palmitoylated. Proc. Natl. Acad. Sci. USA 90: 3675-3679, 1993[Abstract].

13.   McCallum, J. F., A. Wise, M. A. Grassie, A. I. Magee, F. Guzzi, M. Parenti, and G. Milligan. The role of palmitoylation of the guanine nucleotide binding protein G11alpha in defining interaction with the plasma membrane. Biochem. J. 310: 1021-1027, 1995[Medline].

14.   Mileykovskaya, E. I., S. S. Kormer, and W. S. Allison. Significant quantities of endogenous GDP and ADP are present on catalytic sites of the F1-ATPase isolated from M. lysodeikticus in the absence of added nucleotides. Biochim. Biophys. Acta 1099: 219-225, 1992[Medline].

15.   Milligan, G., M. Parenti, and A. I. Magee. The dynamic role of palmitoylation in signal transduction. Trends Biochem. Sci. 20: 181-186, 1995[Medline].

16.   Milligan, G., and C. Unson. Persistent activation of the alpha  subunit of Gs promotes its removal from the plasma membrane. Biochem. J. 260: 837-841, 1989[Medline].

17.   Morre, D. J., and D. M. Morre. Preparation of mammalian plasma membranes by aqueous two-phase partition. Biotechniques 7: 946-958, 1989[Medline].

18.   Mullaney, I., F. M. Mitchell, J. F. McCallum, N. J. Buckley, and G. Milligan. The human muscarinic M1 acetylcholine receptor, when expressed in CHO cells, activates and downregulates both Gqalpha and G11alpha equally and nonselectively. FEBS Lett. 324: 241-245, 1993[Medline].

19.   Mumby, S. M., C. Kleuss, and A. G. Gilman. Receptor regulation of G-protein palmitoylation. Proc. Natl. Acad. Sci. USA 91: 2800-2804, 1994[Abstract].

20.   Navas, P., D. D. Nowack, and D. J. Morre. Isolation of purified plasma membranes from cultured cells and hepatomas by two-phase partition and preparative free-flow electrophoresis. Cancer Res. 49: 2147-2156, 1989[Abstract].

21.   Negishi, M., H. Hashimoto, and A. Ichikawa. Translocation of alpha  subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells. J. Biol. Chem. 267: 2364-2369, 1992[Abstract/Free Full Text].

22.   Ransnas, L. A., and P. A. Insel. Subunit dissociation is the mechanism for hormonal activation of the Gs protein in native membranes. J. Biol. Chem. 263: 17239-17242, 1988[Abstract/Free Full Text].

23.   Ransnas, L. A., P. Svoboda, J. R. Jasper, and P. A. Insel. Stimulation of beta -adrenergic receptors of S49 lymphoma cells redistributes the alpha  subunit of the stimulatory G protein between cytosol and membranes. Proc. Natl. Acad. Sci. USA 86: 7900-7903, 1989[Abstract].

24.   Raymond, J. R., C. L. Olsen, and T. W. Gettys. Cell-specific physical and functional coupling of human 5-HT1A receptors to inhibitory G protein alpha -subunits and lack of coupling to GSalpha . Biochemistry 32: 11064-11073, 1993[Medline].

25.   Rindler, M. J., L. M. Chuman, L. Shaffer, and M. H. Saier. Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK). J. Cell Biol. 81: 635-648, 1979[Abstract].

26.   Rodbell, M. Programmable messengers: a new theory of hormone action. Trends Biochem. Sci. 10: 461-464, 1985.

27.   Salceda, R., G. R. E. M. van Roosmalen, P. A. A. Jansen, S. L. Bonting, and F. J. M. Daemen. Nucleotide content of isolated bovine ROD outer segments. Vision Res. 22: 1469-1474, 1982[Medline].

28.   Shah, B. H., D. J. MacEwan, and G. Milligan. Gonadotrophin-releasing hormone receptor agonist-mediated down-regulation of Gqalpha /G11alpha (pertussis toxin-insensitive) G proteins in alpha t3-1 gonadotroph cells reflects increased G protein turnover but not alterations in mRNA levels. Proc. Natl. Acad. Sci. USA 92: 1886-1890, 1995[Abstract].

29.   Shah, B. H., and G. Milligan. The gonadatropin-releasing hormone receptor of alpha T3-1 pituitary cells regulates cellular levels of both the phosphoinositidase C-linked G proteins, Gqalpha and G11alpha , equally. Mol. Pharmacol. 46: 1-7, 1994[Abstract].

30.   Svoboda, P., G. Kim, M. A. Grassie, K. A. Eidne, and G. Milligan. Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha : analysis of agonist regulation of coexpressed G11alpha species variants. Mol. Pharmacol. 49: 646-655, 1996[Abstract].

31.   Svoboda, P., and G. Milligan. Agonist-induced transfer of the alpha  subunits of the guanine-nucleotide-binding regulatory proteins Gq and G11 and of muscarinic m1 acetylcholine receptors from plasma membranes to a light-vesicular membrane fraction. Eur. J. Biochem. 224: 455-462, 1994[Abstract].

32.   Wedegaertner, P. B., and H. R. Bourne. Activation and depalmitoylation of Gsalpha . Cell 77: 1063-1070, 1994[Medline].

33.   Wedegaertner, P. B., H. R. Bourne, and M. von Zastrow. Activation-induced subcellular redistribution of Gsalpha . Mol. Biol. Cell 7: 1225-1233, 1996[Abstract].

34.   Wedegaertner, P. B., D. H. Chu, P. T. Wilson, M. J. Levis, and H. R. Bourne. Palmitoylation is required for signaling functions and membrane attachment of Gqalpha and Gsalpha . J. Biol. Chem. 268: 25001-25008, 1993[Abstract/Free Full Text].

35.   Wedegaertner, P. B., P. T. Wilson, and H. R. Bourne. Lipid modifications of trimeric G proteins. J. Biol. Chem. 270: 503-506, 1995[Free Full Text].


Am J Physiol Renal Physiol 276(4):F528-F534
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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