©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Factors Determining Specificity of Signal Transduction by G-protein-coupled Receptors
REGULATION OF SIGNAL TRANSFER FROM RECEPTOR TO G-PROTEIN (*)

Motohiko Sato (§) , Ryo Kataoka (§) , Jane Dingus , Michael Wilcox , John D. Hildebrandt , Stephen M. Lanier (¶)

From the (1)Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Among subfamilies of G-protein-coupled receptors, agonists initiate several cell signaling events depending on the receptor subtype (R) and the type of G-protein (G) or effector molecule (E) expressed in a particular cell. Determinants of signaling specificity/efficiency may operate at the R-G interface, where events are influenced by cell architecture or accessory proteins found in the receptor's microenvironment. This issue was addressed by characterizing signal transfer from R to G following stable expression of the adrenergic receptor in two different membrane environments (NIH-3T3 fibroblasts and the pheochromocytoma cell line, PC-12). Receptor coupling to endogenous G-proteins in both cell types was eliminated by pertussis toxin pretreatment and R-G signal transfer restored by reconstitution of cell membranes with purified brain G-protein. Thus, the receptor has access to the same population of G-proteins in the two different environments. In this signal restoration assay, agonist-induced activation of G was 3-9-fold greater in PC-12 as compared with NIH-3T3 -adrenergic receptor transfectants. The cell-specific differences in signal transfer were observed over a range of receptor densities or G-protein concentration. The augmented signal transfer in PC-12 versus NIH-3T3 transfectants occurred despite a 2-3-fold lower level of receptors existing in the R-G-coupled state (high affinity, guanyl-5`-yl imidodiphosphate-sensitive agonist binding), suggesting the existence of other membrane factors that influence the nucleotide binding behavior of G-protein in the two cell types. Detergent extraction of PC-12 but not NIH-3T3 membranes yielded a heat-sensitive, macromolecular entity that increased S-labeled guanosine 5`-O-(thiotriphosphate) binding to brain G-protein in a concentration-dependent manner. These data indicate that the transfer of signal from R to G is regulated by a cell type-specific, membrane-associated protein that enhances the agonist-induced activation of G.


INTRODUCTION

Members of the G-protein-coupled receptor superfamily play a central role in cellular communication mediating the cell response to numerous hormones and neurotransmitters. Via coupling to heterotrimeric guanine nucleotide binding proteins (G),()receptor activation regulates numerous intracellular effector molecules including ion channels, adenylyl cyclases, and phospholipases. Many of these receptors share common signaling pathways that are activated with varying degrees of efficiency by a particular hormone. The specific intracellular signal initiated by agonist and the strength of signal propagation likely depend on many factors, including the relative types and/or amounts of the signal-transducing entities (receptor (R)/G-protein/effector (E))(1, 2, 3, 4, 5, 6, 7, 8) . Additional determinants of signaling specificity include cell architecture and perhaps accessory proteins that regulate events at the R-G or G-E interface.

In attempts to further define determinants of signaling specificity, we focused our efforts on one receptor subfamily, -adrenergic receptors (-AR)(5, 6, 7) . The -AR family consists of three genetically defined receptor subtypes (, , -AR) and shares many properties with other subfamilies of G-protein-coupled receptors in terms of signaling flexibility(9) . Based on results in cells expressing the endogenous receptor gene or in cells stably transfected with receptor subtypes, members of this receptor subfamily couple to phospholipase A(10) , phospholipase C(11) , phospholipase D(12) , calcium flux(13, 14, 15, 16) , Na/H exchangers(17, 18) , p21(19), or adenylyl cyclases(6, 20, 21, 22, 23, 24, 25) . The specific regulation of any of these effector molecules often depends upon the receptor subtype, receptor density, and the environment in which the receptor is operating.

Utilizing the -AR subfamily as a representative subgroup of G-protein-coupled receptors, we have attempted to define how signaling specificity is engineered in the intact cell(5, 6, 7) . Critical determinants of signaling efficiency/specificity may operate at the R-G interface. To address this possibility, we developed a system to evaluate R-G coupling in different membrane environments and to identify factors that may regulate the level of activated G. The present report indicates that the transfer of signal from R to G is regulated by cell-specific membrane-associated proteins that alter the nucleotide binding behavior of G.


EXPERIMENTAL PROCEDURES

Materials

H-Labeled RX821002 (60 Ci/mmol) was obtained from Amersham Corp. [S]GTPS, G/G antisera and H-labeled UK14304 were purchased from Dupont NEN. Tissue culture supplies were obtained from JRH Bioscience (Lenexa, KS). Acrylamide, bisacrylamide, and SDS were purchased from Bio-Rad. Nitrocellulose and polyvinylidene difluoride (Westran) membranes were obtained from Schleicher & Schuell. Propranolol,(-)epinephrine, and pertussis toxin were obtained from Research Biochemicals Inc. The antisera for G/G were provided by Dr. C. Bianchi and Dr. C. Homcy (Cardiac Unit, Massachusetts General Hospital). G antisera was kindly provided by Dr. Eva Neer (Harvard Medical School). Ecoscint A was purchased from National Diagnostics (Manville, NJ). Guanosine diphosphate, thesit (polyoxyethylene-9-lauryl ether), and guanyl-thiodiphosphate were obtained from Boehringer Mannheim.

Membrane Preparations, Radioligand Binding Studies, and Immunoblotting

The rat RG-20 -AR was stably expressed in NIH-3T3 fibroblasts, PC-12 pheochromocytoma cells, or DDT-MF2 smooth muscle cells by cotransfection with the receptor gene in the expression vector pMSV and pNEO, a plasmid that confers G418 resistance(7) . None of the cell lines used for gene transfection expressed the endogenous -AR gene as determined by radioligand binding experiments and RNA blot analysis, as previously described(7) . Cells were grown as monolayers on Falcon Primeria dishes at 37 °C (5% CO) in Dulbecco's modified Eagle's medium with high glucose (4.5 g/liter), supplemented with 10% bovine calf serum (NIH-3T3), 2.5% horse serum, 2.5% bovine calf serum (DDT-MF2) or 5% horse serum, 10% fetal bovine serum (PC-12) plus penicillin (100 units/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml). In some experiments, cells were pretreated with 100 ng/ml pertussis toxin for 18 h (37 °C) prior to membrane preparation. Membranes were prepared and receptor densities and protein concentration determined as previously described(7) . Immunoblotting, densitometric determinations, and Gpp(NH)p-sensitive binding of the selective -AR agonist H-labeled UK14304 (1 nM) were performed as previously described(5, 6) .

Guanine Nucleotide Binding

[S]GTPS binding experiments were performed as described(26) . Briefly, membranes prepared from transfected cells were resuspended in assay buffer (5 mM MgCl, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 1 µM guanosine diphosphate, 1 µM propanolol, 50 mM Tris-HCl, pH 7.4), and the reaction initiated by adding membranes (10 µg in 10 µl) to tubes containing 90 µl of assay buffer containing [S]GTPS (0.2 nM, 1000-1500 Ci/mmol) with or without agonist. Incubation was continued at 24 °C for various times and terminated by rapid filtration through nitrocellulose filters with 4 4 ml of wash buffer (100 mM NaCl, 50 mM Tris-HCl, 5 mM MgCl, pH 7.4, 4 °C). Radioactivity bound to the filters was determined by liquid scintillation counting. Nonspecific binding was defined by 100 µM GTPS or Gpp(NH)p. Several preliminary experiments were performed to establish optimal conditions with respect to time, nucleotide concentration, and buffer (i.e. magnesium and sodium concentration). Under the incubation conditions used, the receptor-mediated increases in [S]GTPS binding in -AR transfectants were PT-sensitive, and the maximal agonist-induced signal was observed at 30-min incubation time points. In the intact PC-12 cell, the -AR couples to both PT-sensitive and PT-insensitive pathways to regulate cellular cAMP levels(7) . Detection of PT-insensitive activation of G-protein in the [S]GTPS binding assay may require different incubation conditions or factors that are lost upon membrane preparation. Receptor coupling was examined in a number of clonal transfectants expressing different receptor densities. All experiments utilized freshly prepared membranes, and receptor density was determined in each membrane preparation using saturating concentrations of H-labeled RX821002, a selective -AR antagonist.

Membrane/G-protein Reconstitution

To eliminate receptor coupling to G-proteins endogenous to the cell, we developed a system in which agonist signal was restored by addition of bovine brain G-protein membranes prepared from PT-treated cells. Based upon earlier studies involving signal reconstitution(27, 28, 29, 30) , several experimental paradigms were evaluated to achieve optimal conditions for reconstitution of the agonist-induced signal. Using the agonist-induced increase in [S]GTPS binding as a readout, we evaluated different preincubation times of membranes and G-protein, different detergent concentrations, and different buffer conditions (Mg/GDP). The standard assay system was as follows. A preincubation mixture was prepared for each assay point. A single assay point consisted of six test tubes, two for total binding, two for nonspecific binding, and two for agonist addition. Nonspecific binding was defined with 100 µM GTPS or Gpp(NH)p. Basal specific binding (without agonist) progressively decreased with increasing concentrations of GDP. Inclusion of GDP (10-100 µM) thus increased the signal to noise ratio and facilitated detection of agonist activation of G. Unless indicated otherwise, the final concentration of GDP was 10 µM. The preincubation mixture consisted of brain G-protein and 60 µg of membranes in 120 µl of buffer A (50 mM Tris-HCl, pH 7.4, 5 mM MgCl, 0.6 mM EDTA) containing 0.005% thesit (polyoxyethylene-9-lauryl ether) and 50 µM GDP. The concentrations of brain G-protein and GDP in the preincubation mixture were five times the final concentration desired in the assay tube. After 1 h of incubation of the preincubation mixture at 4 °C, 20 µl of the mixture was added to the assay tube containing buffer A plus (final concentrations) 150 mM NaCl, 1 mM dithiothreitol, 1 µM propanolol, 50,000 cpm (0.2 nM) [S]GTPS, and agonist, vehicle, or 100 µM GTPS (total volume = 80 µl). Incubation was then continued at 24 °C for various times, and the reaction was terminated by rapid filtration through nitrocellulose filters (Schleicher & Schuell, BA85) with 4 4-ml washes (50 mM Tris-HCl, 5 mM MgCl, pH 7.4, 4 °C). Radioactivity bound to the filters was determined by liquid scintillation counting. High affinity, Gpp(NH)p-sensitive binding of the -AR agonist H-labeled UK14304 in the signal restoration system was determined in a similar manner using 50 µg of membrane protein and 50-300 nM brain G-protein with exclusion of sodium and GDP from the preincubation mixture.

Preparation of Membrane Extract

PC-12 or NIH-3T3 membrane preparations were solubilized with 1% sodium cholate (detergent:protein ratio of 2:1) in membrane buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl, 0.6 mM EDTA) by incubation at 4 °C for 30 min. The solubilized material was then centrifuged at 100,000 g for 1 h, and the supernatant was used as the membrane extract. To determine the effect of membrane extract on [S]GTPS binding to brain G-protein, membrane extracts (6-12 µg of protein) were preincubated with brain G-protein, and the reaction was initiated as described above. In some experiments, the membrane extract was concentrated by centrifugation using YM10 centricon tubes (AMICON).

Purification of Bovine Brain G-protein

Bovine brain G-protein heterotrimer was purified by a modification (28, 31) of the technique of Sternweis and Robishaw(32) . Based on subsequent fractionation, immunoblot analysis and protein sequencing, the brain G-protein preparation was approximately 63% G, 4% G, 16% G, 16% G, 1% G. The heterotrimeric G-protein preparation was isolated in its GDP-liganded form. Greater than 80% of the heterotrimer was functional based on the amount of [S]GTPS binding expected from protein determinations.


RESULTS

G-protein Activation by Epinephrine in Receptor Transfectants

The cell response to -AR activation in NIH-3T3 versus PC-12 transfectants differs in events at both the R-G and G-E interface. In NIH-3T3 transfectants, receptor activation inhibits adenylyl cyclase by coupling to PT-sensitive heterotrimeric G-proteins. However, in PC-12 transfectants, the receptor couples to PT-sensitive and PT-insensitive pathways that elicit negative (PT-sensitive) or positive (PT-insensitive) effects on adenylyl cyclase activity(5, 7) . The cell type-specific effects of receptor activation on cAMP in PC-12 versus NIH-3T3 cells are described elsewhere and reflect, in part, cell-specific expression of different adenylyl cyclase enzymes (7).()The present report indicates that cell-specific signaling events also occur proximal to effector activation as reflected in agonist-induced activation of G-protein in the two cell types.

Receptor-mediated activation of guanine-nucleotide binding proteins was determined by measuring the increase in [S]GTPS binding elicited by the -AR agonist epinephrine in membrane preparations of the receptor transfectants (Fig. 1). The magnitude of the increase in [S]GTPS binding elicited by epinephrine was related to receptor density, and the signal was time dependent with maximal responses observed at 30 min under these incubation conditions (Fig. 1).()The agonist-induced signal was determined in both NIH-3T3 and PC-12 -AR transfectants expressing a range of receptor densities. Epinephrine elicited a greater degree of G-protein activation in PC-12 as compared with NIH-3T3 -AR transfectants over the entire range of receptor densities (800-9000 fmol/mg membrane protein) (Fig. 1B).


Figure 1: Increase in [S]GTPS binding elicited by agonist in NIH-3T3 and PC-12 -AR transfectants. Clonal receptor transfectants were generated and receptor densities determined as described under ``Experimental Procedures.'' In each experiment, the level of [S]GTPS binding was determined in the presence or absence of epinephrine (10 µM) for various incubation times (30 min in B) at 24 °C. A, receptor density (fmol/mg): NIH-3T3, 1400; PC-12, 1100. Data presented in A are representative of three to five separate experiments with different clonal cell lines. Data in B are presented as the mean ± S.E. of four experiments with different membrane preparations of the individual clonal cell lines. A, basal [S]GTPS binding at 2, 5, 10, 30, and 60 min; NIH-3T3, 0.57, 0.88, 1.37, 1.66, and 2.50; PC-12, 0.98, 1.20, 1.91, 3.29, and 4.27.



Receptor-mediated activation of G-protein was also evaluated in another cell type stably expressing the receptor protein. In DDT-MF2 -AR transfectants, the epinephrine-induced increase in [S]GTPS binding was similar to that observed in NIH-3T3 transfectants (Fig. 2). In each cell type, the agonist-mediated signal required receptor expression and was blocked by pretreatment of cells with PT, implicating a subgroup of G-proteins involved in signal transfer (Fig. 2). Although the agonist-induced activation of G was greater in PC-12 versus NIH-3T3 and DDT-MF2 -AR transfectants, the population of receptors exhibiting high affinity for agonist (the R-G-coupled state) was 2-4-fold higher in the latter two cell types compared with PC-12 cells (Fig. 2), indicating that two separate events are required for productive R-G coupling: R-G interaction and G-activation (subunit dissociation and guanine nucleotide exchange). Experiments were then designed to investigate the mechanism of the cell-specific differences in G activation by agonist.


Figure 2: Comparison of agonist-induced activation of G and Gpp(NH)p-sensitive binding of the selective -AR agonist H-labeled UK14304 in NIH-3T3, DDT-MF2, and PC-12 -AR transfectants. Membranes were prepared from control or PT-treated cells (PT) as described under ``Experimental Procedures.'' Aliquots of the membrane preparations were then evaluated for high affinity agonist binding and agonist-mediated effects on [S]GTPS binding. Results are presented as the mean ± S.E. of four experiments. Receptor density (fmol/mg): NIH-3T3, 5200; PC-12, 4550; DDT-MF2, 4000. Gpp(NH)p = 100 µM; H-labeled UK14304 = 1 nM; epinephrine = 10 µM. A, basal [S]GTPS binding (fmol): NIH-3T3, 1.61 ± 0.105; PT+, 0.27 ± 0.045; PC-12, 2.12 ± 0.14; PT+, 0.47 ± 0.08; DDT-MF2, 1.56 ± 0.1; PT+, 0.19 ± 0.04. B, specific binding of H-labeled UK14304 (1 nM) in control cells (without pertussis toxin pretreatment) in the absence of Gpp(NH)p: 859 ± 183 cpm, NIH-3T3; 289 ± 32 cpm, PC-12; 489 ± 80 cpm, DDT-MF2. Data are presented as the total amount of specific ligand binding that is inhibited by Gpp(NH)p. Counting efficiency = 50%. In A and B, DDT refers to DDT-MF2 cells.



Cell Type-specific Expression of G

Within the family of PT-sensitive G-proteins, NIH-3T3 fibroblasts and PC-12 cells express G, G and G, G, G, G, respectively.()Differences in the type or amount of G-protein expressed in the two cell types may contribute to augmented signal transfer in PC-12 transfectants either by enhanced R-G coupling efficiency or the GTP binding properties of the G subunit itself.

The apparent differences in signal transfer between NIH-3T3/DDT-MF2 cells and PC-12 cells were not due to greater levels of G and G per µg of membrane protein in PC-12 cell membranes (Fig. 3A). The levels of G per µg of membrane protein were actually lower in PC-12 cell membranes compared with the other two cell types. To determine if the expression of G in PC-12 but not NIH-3T3 transfectants accounted for the cell type difference in signal transfer, we re-analyzed R-G coupling after stable expression of G in NIH-3T3 fibroblasts. In NIH-3T3 fibroblasts stably cotransfected with the receptor and G(6) , the agonist-induced signal was augmented relative to cells transfected with pMSV.-AR alone (Fig. 3B). However, despite the expression of G in NIH-3T3 transfectants at levels 7-fold higher than the amount of immunoreactive G found in PC-12 cells (Fig. 3B, inset), the effect of epinephrine on [S]GTPS binding in NIH-3T3 receptor/G cotransfectants was still less than that observed in PC-12 transfectants. These data indicate a more productive coupling of R and G in PC-12 cells.


Figure 3: G-protein expression in NIH-3T3, DDT-MF2, and PC-12 cell membranes and agonist activation of G-proteins in NIH-3T3 fibroblasts cotransfected with the -AR and G. A, identification of G/G or G/G in NIH-3T3, DDT-MF2, and PC-12 membranes. B, agonist-induced activation of G-proteins in NIH-3T3 and PC-12 -AR transfectants and in NIH-3T3 -AR/G cotransfectants. Epinephrine = 10 µM. Immunoblot-membrane protein (50 µg for each cell type, 20 µg of brain) from different cell types were electrophoresed and transferred to Westran membranes; the blots were incubated with antisera selective for G/G or G/G in A or G in B (inset) (7). The doublet in the G/G immunoblot of PC-12 membranes in A is due to antibody cross-reactivity with G. Receptor density (fmol/mg): NIH-3T3, 1100; NIH-3T3/G, 1200; PC-12, 1200.



Signal Reconstitution with Purified Brain G-protein Heterotrimer

The results of the preceding experiments did not entirely eliminate a role for cell-specific expression of G types in the differences in R-G coupling in the two cell types. Therefore, the transfer of signal from R to G was further characterized by evaluating the ability of each cell membrane preparation to support signal restoration by exogenous heterotrimeric G-proteins. In this series of experiments, receptor coupling to endogenous G-proteins was eliminated by pertussis toxin treatment of the cells prior to membrane preparation. Pertussis toxin-treated membranes were reconstituted with a purified preparation of brain heterotrimeric G-protein, and thus the receptor accesses the same population of G-proteins. As shown in Fig. 4, the agonist-induced response (eliminated by PT) was restored by preincubation of membranes and brain G-protein. The agonist-induced increase in [S]GTPS binding was dose dependent, inhibited by the -AR antagonist rauwolscine, and required receptor expression as it was not observed in control pMSV.neo-transfected cells (Fig. 4).


Figure 4: Agonist-induced activation of G-protein in the membrane/brain G-protein reconstitution system using PC-12 -AR transfectants. Membranes were prepared from cells pretreated with pertussis toxin and reconstituted with 25 nM bovine brain G-protein as described under ``Experimental Procedures.'' A, agonist-induced activation of G-protein in membrane preparations from cells transfected with pMSV.-AR or resistance plasmid alone in the presence and absence of added G-protein. Data are presented as the mean ± S.E. of four experiments using different membrane preparations. B, effect of increasing epinephrine concentrations on [S]GTPS binding in the presence and absence of the -AR antagonist rauwolscine. Receptor density = 5400 fmol/mg. The results are representative of three experiments using different clonal cell lines and are expressed as the percent of the epinephrine-induced increase in [S]GTPS binding at 100 µM agonist ([S]GTPS binding: basal = 0.81 fmol, epinephrine (100 µM) = 3.45 fmol).



The signal restoration system was then used to compare NIH-3T3 and PC-12 transfectants in terms of their ability to support the agonist signal. As is the case in non-PT-treated membranes, the receptor-mediated activation of G-protein in the signal restoration system was up to 8-fold greater in PC-12 versus NIH-3T3 transfectants ( Fig. 5and Fig. 6), and the augmented signal transfer in PC-12 transfectants actually occurred with a 2-4-fold lower amount of receptors (relative to NIH-3T3 fibroblasts) existing in the R-G-coupled state (Fig. 5). Reconstitution of high affinity Gpp(NH)p-sensitive binding in NIH-3T3 transfectants indicates that exogenous G-protein has access to the receptor in both cell types and that the transfer of signal from R to G is more efficient in PC-12 transfectants. Further analysis of signal transfer in the two cell types indicated that the cell-specific differences were observed over a range of heterotrimer or magnesium concentrations (Fig. 6) as well as in membrane preparations that were washed with 1 M KCl to remove proteins loosely associated with the cell membranes.()


Figure 5: Comparison of agonist-induced activation of G and Gpp(NH)p-sensitive binding of the selective -AR agonist H-labeled UK14304 in NIH-3T3 and PC-12 -AR transfectants in the membrane/G-protein reconstitution assay. Membranes were prepared from pertussis toxin-treated cells and reconstituted with 25 (A) or 50 and 300 (B) nM brain G-protein as described under ``Experimental Procedures.'' A, basal [S]GTPS binding (fmol) ranged from 0.75 to 1.24 in NIH-3T3 transfectants and from 1.19 to 2.14 in PC-12 transfectants. Data are presented as the mean of duplicate determinations using different clonal cell lines. Experiments were repeated twice in each transfectant with similar results. Epinephrine = 10 µM. See legend to Fig. 2 for additional details.




Figure 6: Agonist-induced activation of G-protein in the membrane/brain G-protein reconstitution system using PC-12 and NIH-3T3 -AR transfectants and the effect of magnesium and G-protein concentration. Membranes were prepared from cells pretreated with pertussis toxin to eliminate receptor coupling to endogenous G and then reconstituted with heterotrimeric G-protein (25 nM in A) purified from bovine brain. Epinephrine = 10 µM. Receptor density (fmol/mg): NIH-3T3, 5600 (A) and 1100 (B); PC-12, 5100 (A) and 950 (B). Data are presented as the mean of duplicate determinations and are representative of two (A) or five (B) separate experiments using different membrane or G-protein preparations. GDP concentration in A = 30 µM and in B = 100 µM. A, basal [S]GTPS binding (fmol) at 0.3, 1, 3, and 10 mM MgCl: NIH-3T3, 0.08, 0.2, 0.24, 0.28; PC-12, 0.09, 0.31, 0.67, 0.81.



Effect of Cell Membranes and Membrane Extracts on [S]GTPS Binding to Purified Brain Heterotrimeric G-protein

The results of the signal reconstitution experiments suggest the existence of a membrane factor in PC-12 or NIH-3T3 cells that alters guanine nucleotide binding to heterotrimeric G-protein. This issue was addressed by analysis of the guanine nucleotide binding behavior of purified brain G-protein alone or following preincubation of brain G-protein with membrane preparations or membrane extracts from nontransfected PC-12 or NIH-3T3 cells. In each experiment, [S]GTPS binding was measured in the brain G-protein preparation and the membrane preparation/extract alone and then compared with the amount of nucleotide bound when the two preparations were co-incubated. In the absence of any ``regulatory factors,'' the [S]GTPS binding in the co-incubations would be additive and represent the sum of nucleotide binding in either preparation alone. However, in the presence of PC-12 membrane preparations, [S]GTPS binding to purified brain G-protein was increased by 50-120% above the value expected from summation of nucleotide binding in the two preparations alone (Fig. 7). The greater than additive effect was augmented at lower GDP concentrations and was not observed when purified brain G-protein was incubated with PC-12 cytosol.()In contrast, incubation of purified brain G-protein with NIH-3T3 membrane preparations resulted in a level of nucleotide binding that was close to additive, relative to results obtained with brain G-protein or NIH-3T3 membranes alone.


Figure 7: [S]GTPS binding to brain G-protein in the presence and absence of cell membrane preparations. Membranes were prepared from nontransfected PC-12 or NIH-3T3 cells as described under ``Experimental Procedures.'' [S]GTPS binding (2 nM) to brain G-protein (12.5 nM) was determined in the absence and presence of 10 µg of cell membrane protein. Brain G-protein and membrane were preincubated for 1 h at 4 °C, and the binding reaction was initiated as described under ``Experimental Procedures.'' Data are presented as the mean of duplicate determinations and are representative of three experiments with different membrane preparations.



As indicated above, the cell type-specific differences in signal transfer were maintained following ``stripping'' of peripheral membrane proteins (1 M KCl wash), suggesting that the regulatory activity is tightly associated with the membrane. Subsequent experiments indicated that the regulatory activity was extracted from the membrane by detergent solubilization with sodium cholate. PC-12 membrane extract increased the binding of [S]GTPS binding to brain G-protein by 65-75%, whereas NIH-3T3 membrane extract was without effect (Fig. 8A). The PC-12 membrane extract also augmented basal and receptor-mediated activation of G-protein in NIH-3T3 -AR transfectants using the signal reconstitution assay. Membrane extracts prepared from NIH-3T3 fibroblasts were without effect in this system. The effect of PC-12 membrane extract (15% increase) on receptor-mediated activation of G in NIH-3T3 -AR transfectants was less than the effect of the extract on [S]GTPS binding to brain G-protein alone in the solution-phase assay, likely reflecting the experimental conditions of the two different assay systems.


Figure 8: Effect of detergent-solubilized membrane extracts on [S]GTPS binding to brain G-protein. [S]GTPS binding (2 nM) to brain G-protein (12.5 nM) was determined in the absence and presence of membrane extract (A, 5.5 µg of protein). Membrane extracts were prepared from nontransfected PC-12 or NIH-3T3 by membrane solubilization with sodium cholate as described under ``Experimental Procedures.'' Brain G-protein and membrane extract or vehicle were preincubated for 1 h at 4 °C, and the binding reaction was initiated as described under ``Experimental Procedures.'' A, concentrate: the membrane extract was concentrated 5-fold by centrifugation in a centricon concentrater with a 10,000 molecular size exclusion membrane and then diluted to the original volume with solubilization buffer prior to assay. 95 °C: an aliquot of the retentate was placed in a boiling water bath for 5 min and cooled to 4 °C before preincubation with brain G-protein. filtrate: flow-thru fraction obtained by centrifugation of the membrane extract in a centricon concentrater with a 10,000 molecular size exclusion membrane. The protein concentration in the filtrate was below detectable levels, and thus the amount of filtrate used was equivilant to the volume of unconcentrated membrane extract added to the assay. In A, data are presented as the mean ± S.E. of three experiments and represent the increase above the summed value of nucleotide binding in extract and G-protein preparation alone. The effect of NIH-3T3 membrane extract was not statistically different from control. In B, a constant detergent concentration was maintained for each assay point. Data are presented as the mean of duplicate determinations from two separate experiments, and the values in the open and closedsquares correspond to increases above the summed value of nucleotide binding in extract and G-protein preparation alone. The GDP concentration was 1 µM in both A and B. Under the experimental conditions used, the amounts of [S]GTPS bound in PC-12 or NIH-3T3 membrane extracts alone were 3-4 and 1-2 fmol, respectively. In such assay conditions, 25 fmol of [S]GTPS was bound to purified brain G-protein alone.



The stimulatory action of the detergent-solubilized PC-12 membrane preparation was related to the protein concentration of the extract (Fig. 8B). The regulatory activity was found in the retentate following centrifugation of the PC-12 membrane extract in a YM10 membrane (10,000 molecular size exclusion) centricon, and the stimulatory effect of PC-12 membrane extract was eliminated by heating of the extract at 95 °C for 5 min (Fig. 8A). Both observations suggest that the ``regulatory factor'' is a protein and not a lipid/nucleotide derivative of low molecular size (Fig. 8A).


DISCUSSION

A major determinant of signaling specificity for G-protein-coupled receptors is the cell-specific expression of the primary signaling entities, R, G, and E. A secondary line of signaling specificity lies at the R-G or G-E interface, where these interactions may be influenced by cell architecture, stoichiometry, and accessory proteins that regulate signal transfer from R to G or G to E. Through the use of various experimental systems, we are attempting to define the relative importance of these factors in determining the efficiency and selectivity of receptor-effector coupling.

NIH-3T3 and PC-12 cells provide an interesting context in which to address the above issues. Both cell types express distinct and common signaling entities and differ in their cell architecture. Both the - and -AR subtypes differ in their coupling to adenylyl cyclase when expressed in the two cell types(6) . The present report indicates that the PC-12 and NIH-3T3 -AR transfectants also differ in the efficiency/magnitude with which the agonist-occupied receptor activates both endogenous and exogenous heterotrimeric G-proteins. In PC-12 transfectants, a neuron-like cell line, the receptor appears to transfer the agonist-occupation event to G-protein activation with greater efficiency than it does when expressed in NIH-3T3 fibroblasts or DDT-MF2 smooth muscle cells.

Although R, G, and E are the major entities in the signaling process, other putative regulatory proteins may participate either indirectly by structural support(33, 34) , compartmentalization(35) , and signal cross-talk (36) or directly by inhibiting or stimulating R-G or G-E coupling events(37, 38, 39, 40, 41, 42, 43, 44, 45) . The GTP-bound/GDP-bound state of p21-related monomeric G-proteins is regulated by specific proteins that stimulate dissociation of bound GDP or GTPase activity. Analogous functions for heterotrimeric G-proteins may be subserved by receptor, G, or effector as indicated by experiments in which purified R, G, or E is reconstituted in phospholipid vesicles. However, a role for additional proteins (i.e. arrestin, phosducin, neuromodulin, kinases) in regulating cell signaling by G-protein-coupled receptors may be lost in reconstitution experiments using purified proteins and could only be observed in the natural cell environment as described in the present report.

The augmented response to agonist in PC-12 versus NIH-3T3 transfectants (in the absence of PT pretreatment) may be due to expression of different types/amounts of PT-sensitive G or G. Thus, the more efficient response in PC-12 membranes may reflect either a preferred and more effective coupling of the receptor to a heterotrimer (i.e. G) not found in NIH-3T3 fibroblasts or the GTP/GDP binding properties of the G activated by the receptor in the two cell types. However, despite 7-fold greater expression of G in NIH-3T3 cells relative to the levels of immunoreactive G expressed in PC-12 cells, the epinephrine-induced signal in NIH-3T3 receptor/G cotransfectants did not reach that elicited by the agonist in PC-12 transfectants. These data and the lower amount of receptors actually coupled to G (high affinity agonist binding) in PC-12 versus NIH-3T3 or DDT-MF2 membranes suggest that the transfer of signal from R to G is regulated by other entities in the receptor's microenvironment. Such a possibility is supported by the results of the signal reconstitution system in which the receptor is coupling to the same population of G within the two membrane environments.

The signal restoration system involves 1) inactivation of receptor coupling to endogenous G-proteins by pertussis toxin pretreatment and 2) restoration of signal by addition of a purified brain G-protein preparation. The approach is possible because agonist activation of G-protein in both cell types is blocked by ADP-ribosylation of G-proteins with pertussis toxin. The signal restoration system potentially allows the identification of unknown entities that regulate the transfer of signal from R to G or G to E. In this system, the transfer of signal from R to G occurs much more efficiently when the receptor is functioning in the PC-12 versus the NIH-3T3 membrane environment. Furthermore, the degree of agonist activation of G was ``dissociated'' from the amount of receptor existing in the R-G-coupled state (high affinity agonist binding). Thus, Gpp(NH)p-sensitive binding of the selective -AR agonist H-labeled UK14304 in NIH-3T3 transfectants is 2-4-fold higher than that observed in PC-12 transfectants in both PT-untreated membranes and the membrane/brain G-protein reconstitution system. These data indicate that factors other than the presence or absence of a particular G heterotrimer contribute to the difference in signal transfer in the two cell types and that the transfer of signal from R to G is regulatable. Indeed, the results of subsequent experiments indicated the existence of a cell type-specific membrane-associated protein that directly activates G and influences the propagation of agonist-induced signals.

A working hypothesis is that signaling efficiency/specificity is determined in part by proteins found in the receptor's microenvironment, which together with R, G, and E contribute to the formation of a signal transduction complex at the cytoplasmic face of the receptor. The signal transduction network for this system may parallel that used by receptors with a single membrane span motif where binding of agonist initiates a series of protein-protein interactions dependent on protein phosphorylation. This hypothesis is consistent with data suggesting the existence of multimeric G-protein subunit complexes and the isolation of receptor or G-protein subunits together with some effectors(46, 47, 48, 49, 50) . Detailed reconstruction of the receptor's microenvironment and identification of complexed molecules will provide insight as to mechanisms of signaling specificity and may allow targeting of therapeutic agents to the R-G or G-E interface as opposed to the receptor's hormone binding site.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants NS24821 (to S. M. L.) and DK37219 (to J. D. H.) and the Council for Tobacco Research CTR 2235 (to S. M. L.). This paper is the fourth in the series "Factors Determining Specificity of Signal Transduction by G-protein-coupled Receptors." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work and are visiting scientists from Asahikawa Medical College, Asahikawa, Japan.

To whom correspondence should be addressed: Dept. of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2574; Fax: 803-792-2475.

The abbreviations used are: G, G-protein; R, receptor; E, effector molecule; -AR, adrenergic receptor; PT, pertussis toxin; GTPS, guanosine 5`-3-O-(thio)triphosphate; Gpp(NH)p, guanosine 5`-(,-imido)triphosphate.

M. Sato, E. Duzic, and S. M. Lanier, unpublished observations.

A portion of these studies was presented in preliminary form (Tian, W. N., Deth, R., Lanier, S. M., and Duzic, E. (1992) FASEB J.6, 2423).

E. Duzic and S. M. Lanier, unpublished observations.

M. Sato, R. Kataoka, J. Hildebrandt, and S. M. Lanier, unpublished observations.

M. Sato, J. Hildebrandt, and S. M. Lanier, unpublished observations.


ACKNOWLEDGEMENTS

We appreciate the discussion of Dr. Emir Duzic, who generated the immunoblot shown in Fig. 3A. We thank Bronwyn Tatum for assistance in the preparation of bovine brain G-protein.


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