©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Coupling of -Adrenergic Receptor to Hematopoietic-specific G Proteins (*)

Dianqing Wu (§) , Yanan Kuang (1), Yanping Wu , Huiping Jiang

From the (1)Department of Pharmacology and Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The coupling of the -adrenergic receptor (AR) to the subunits of the Gq class of G proteins was investigated in a cotransfection system. COS-7 cells cotransfected with the -AR cDNA and the G or G cDNA showed marked norepinephrine-induced increases in accumulation of inositol phosphates in a concentration-dependent manner. However, cells cotransfected with the cDNA encoding Gq, G, or G instead of G gave no ligand-dependent activation of phospholipase C (PLC). The facts that the -AR agonist isoprenaline can also induce activation of PLC in cells coexpressing -AR and G and that the -AR-specific antagonist propranolol can block norepinephrine-induced activation of PLC in these cotransfected cells further indicate that it is the -AR that mediates the activation of phospholipase C in these cotransfected cells. To test the possibility of involvement of G, a G antagonist, G3 mutant with substitution of a Ser residue for the C-terminal Cys residue, was used because this protein, when expressed in COS-7 cells, can inhibit only G-mediated but not G-mediated activation of PLC. The result that the G3 mutant could not inhibit -adrenergic receptor-mediated activation of PLC in cells cotransfected with the G cDNA suggests that G is unlikely to be a major mediator of -adrenergic receptor-induced activation of PLC. Thus, we conclude that the -adrenergic receptor can specifically couple to G and G, but not to Gq, G, or G to activate PLC.


INTRODUCTION

G protein-mediated signal transduction pathways are involved in a wide range of biological processes, including metabolism, neuronal activities, some of the sensory processes and hematopoietic functions, etc. The signal transducing cascades are initiated with interactions of ligands with receptors, followed by specific interactions between ligand-bound receptors and G proteins and subsequent activation of effectors by activated G proteins(1, 2) . Molecular cloning has revealed a great deal of diversity in the molecular natures of receptors, G proteins and effectors(3) . The intriguing question is how the signal transduction pathways are organized so that they can function properly and smoothly in order to meet different needs in a variety of cellular systems. The tissue- or cell type-specific expression of the receptors, G proteins, and effectors and selectivity in their interactions are the key factors in organization of the signal transduction networks. For instance, as many as five G subunits, including Gq, G, G, G, and G, have been identified as the members of the Gq class(3) . Gq and G were found in almost all the cells and tissues that have been examined(4, 5) . However, expression of G, G, and G is restrictive: G was found most abundant in the kidney and lung(6) , whereas G(6) and G(6) were only found in hematopoietic cells. Although all the G subunits of the Gq class can activate phospholipase C (PLC)()(7, 8, 9, 10, 11, 12, 13, 14) , they show different specificity in coupling to receptors. Although -AR can couple to all the G subunits of the Gq class to activate PLC(11) , the interleukin-8 receptor can couple only to G, G, and G, but not to Gq or G(15) . The similar results were also observed for the C5a and formylmethionylleucylphenylalanine receptors: these two receptors can couple to G, but not to Gq and G(16) .

Norepinephrine and epinephrine transduce their biological information through a group of cell surface receptors, the adrenergic receptors (AR). These receptors can be divided into three classes, , , and , based on the pharmacological and molecular cloning studies, and multiple subtypes exist in each class(17) . Most of the studies that used the in vivo systems or transfected cells indicated that the -ARs couple to the G subunits of the Gq class to activate PLC(11) ; the -ARs couple to the Gi proteins to inhibit adenylyl cyclase, and the -ARs couple to the Gs proteins to activate adenylyl cyclase(17, 18) . However, a few other studies suggested that the selectivity in the interactions of these ARs with different G proteins is rather promiscuous in certain systems. For example, two reports revealed that -AR could stimulate PLC activity in turkey erythrocytes in a cAMP-independent pathway(19, 20) .

In the present study we investigated whether -AR can couple to any of the G subunits of the Gq class. We found that -AR can specifically couple to G and G, but not Gq, G, or G in the cotransfection assay system.


EXPERIMENTAL PROCEDURES

Cell Culture, Transfection, and Enzyme Assays

COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum under 5% CO at 37 °C. For transfection, COS-7 cells were seeded into 12-well plates at a density of 1 10 cells/well the day before transfection. The media were removed the next day, and 0.5 ml of Opti-MEM (Life Technology, Inc.), containing 3.3 µg of lipofectamine (Life Technology, Inc.) and 1 µg of plasmid DNA, was added to each well. Five hours later the transfection media were replaced by the culture media. Then the cells were labeled with 10 µCi/ml of myo-[2-H]inositol or [H]adenosine the following day, and the levels of inositol phosphates or cAMP were determined 1 day later as described previously(10) . All cDNAs used in this study were constructed in the pCMV expression vector(10) .

Binding Assays

COS-7 cells in 12-well plates were cotransfected with the cDNAs encoding the -AR and one of the subunits of the Gq class. After 48 h, the cells were washed with phosphate-buffered saline and incubated with varying amounts of [H]propranolol (30 Ci/mmol, DuPont NEN) in phosphate-buffered saline for 40 min at 4 °C. Then the cells were washed three times with ice-cold phosphate-buffered saline and lysed in 0.5 ml of 0.2 N NaOH. Aliquots of 0.1 ml were taken for counting in a scintillation counter. The nonspecific binding was determined by measuring binding of [H]propranolol to nontransfected cells. The number of specific binding sites were determined by the Scatchard analysis.


RESULTS AND DISCUSSION

-AR was found to stimulate the release of inositol phosphates from the turkey erythrocyte membranes through cAMP-independent and pertussis toxin-insensitive pathways(19, 20) . To determine the signal transduction pathway for this phenomenon, we tested whether -AR can couple to any of the G subunits of the Gq class of G proteins in the cotransfection assay. We found that cells transfected with the -AR cDNA showed little ligand-induced accumulation of IPs (Fig. 1), suggesting that -AR cannot activate endogenous Gq/11 (COS-7 cells contain endogenous Gq and G, but not G, G, or G(10, 11) . Cotransfection of the cells with the Gq, G (Fig. 1) or G (data not shown) cDNA did not lead to ligand-induced activation of PLC either. This is consistent with the previous results that -AR was a poor activator of Gq, and its specific agonists hardly stimulated the release of IPs in most systems (17, 21) where Gq and G are usually expressed. However, when cells were cotransfected with cDNAs encoding -AR and G, marked increases in ligand-induced accumulation of IPs were found. The concentration-dependent curve indicates that the EC is about 2 µM of norepinephrine (Fig. 1). In addition, cotransfection with the G cDNA gave the same result, suggesting -AR can also couple to G (data not shown). We have demonstrated previously that in this cotransfection system recombinant proteins are usually expressed at levels 3-10-fold higher than endogenous proteins, if there are any(10, 11, 15, 22, 23) , and that cells transfected with the G cDNA expressed G at a level about 3-fold higher than endogenous Gq/11(15, 22) . Furthermore, the numbers of -AR on cells transfected with different cDNAs also remain constant (230 fmol/10 cells). Therefore, all the results indicated that -AR may specifically couple to G and G, but not to Gq, G, or G.


Figure 1: Coupling of -AR to G subunits of the Gq class. COS-7 cells were cotransfected with the -AR cDNA and the cDNA encoding G, G, Gq, or the control -galactosidase (LacZ). The levels of inositol phosphates were determined 20 min after addition of norepinephrine.



To confirm that norepinephrine-induced activation of G is mediated by -AR, we used the -AR-specific agonist isoprenaline. As shown in Fig. 2, 2 µM isoprenaline stimulated accumulation of IPs in cells coexpressing -AR and G. The same amount of isoprenaline showed little effect on cells expressing -AR or G alone, nor did it show any effect on cells cotransfected with the Gq or G cDNA and the -AR cDNA (data not shown). Furthermore, the -AR-specific antagonist propranolol (10 µM) could inhibit norepinephrine-induced activation of PLC in the cells coexpressing G and -AR. Since -AR is known to activate adenylyl cyclases through the Gs proteins, increases in the cAMP levels may indirectly activate the recombinant G protein. To test this possibility, we added forsklin to the cells transfected with the cDNA encoding G. Forsklin showed little effect on the levels of IPs (data not shown), suggesting that -AR-induced activation of G is not mediated by adenylyl cyclases. We also excluded the possibility that coexpression of G may change G protein-coupling specificity of -AR, because -AR can still stimulate accumulation of cAMP in the presence of G (data not shown). Therefore, norepinephrine-dependent activation of PLC in cells coexpressing G and -AR is -AR-specific and cAMP-independent.


Figure 2: Coupling between -AR and G. COS-7 cells were transfected or cotransfected with cDNAs encoding G, LacZ, and/or -AR as indicated in the figure. Ligand-induced accumulation of inositol phosphates was determined 20 min after addition of norepinephrine (Nor, 2.5 µM), isoprenaline (Iso, 2 µM), and/or propranolol (Pro, 10 µM), as indicated.



We have observed previously that COS-7 cells cotransfected with G and G showed a synergistic increase in the level of IPs over those transfected with G or G alone(22) . Thus, it is possible that -AR-mediated activation of PLC in cells cotransfected with the G cDNA may result from the synergistic effect between G and the G subunits that are released from -AR-activated Gs proteins. To test this possibility, we used a dominant negative inhibitor of G, G3CS, with substitution of a Ser residue for the C-terminal Cys residue. We have previously demonstrated that G3CS could inhibit C5a- or formylmethionylleucylphenylalanine-induced activation of PLC , which is mediated by G; but G3CS did not have any effect on m1-muscarinic receptor-mediated activation of PLC, which is mediated by the Gq proteins.()In this report we demonstrated that G3CS was also able to inhibit interleukin-8-induced activation of PLC (Fig. 3A), which is mediated by G (15) without affecting m1-muscarinic receptor-mediated effect (Fig. 3B). These findings indicate that G3CS can serve as a specific antagonist for G-mediated effects. To test whether G is directly involved in -AR-mediated activation of PLC, COS-7 cells were cotransfected with the G and -AR cDNAs and the G3CS cDNA. As shown in Fig. 3C, coexpression of G3CS had no effect on norepinephrine-induced activation of PLC in cells coexpressing -AR and G, suggesting that the G subunits released from Gs do not play a significant role in -AR-mediated activation of PLC and that -AR directly couples to G.


Figure 3: Effects of G on -AR-mediated activation of PLC. COS-7 cells were cotransfected with various cDNAs, including -galactosidase (LacZ), G3CS, G, and -AR, as indicated in the figure. Norepinephrine-induced accumulation of IPs was determined 20 min after addition of ligand (2 µM of norepinephrine).



We have provided clear evidence by the cotransfection assay for the notion that -AR couples to G. The next question is whether the coupling exists in natural systems. Thus, we tested whether isoprenaline can induce accumulation of IPs in THP-1 cells. THP-1 is a human promonocytic leukemia cell line, and -AR on these cells has been well characterized(24, 25) . This cell line also contains G(6) . We found that isoprenaline (10 µM) clearly stimulated accumulation of inositol phosphates in THP-1 cells (Fig. 4), thus providing evidence for the natural occurrence of the coupling between -AR and G. In addition, -AR was previously found to stimulate PLC activity in the erythrocytes(19, 20) . Since both G (6) and -AR (19, 20) were found in the erythrocytes, this result may provide another example for -AR-mediated activation of PLC in natural systems(19, 20) .


Figure 4: -AR-induced activation of PLC in THP-1 cells. THP-1 cells were labeled with [H]inositol for 1 day and then the levels of inositol phosphates were determined 30 min after addition of isoprenaline (10 µM). The dark bars represent cells that were treated with pertussis toxin (500 µg/ml) for 4 h immediately before addition of the ligand. The asterisks represent p < 0.01.



G, G, and -AR were all found in a variety of hematopoietic cells. G was detected in many erythroids, T cell and myeloid lineage(6) , whereas G was detected in a number of B cell as well as myeloid lineages(26) . -AR was also found in many T and B cells as well as myeloid cells(27, 28, 29, 30, 31, 32, 33) . The specific coupling of -AR to G and G suggests that norepinephrine and epinephrine may not only activate adenylyl cyclases, but also activate PLC in many of the hematopoietic systems. In addition, our findings may also explain some of the -AR-mediated effects in certain hematopoietic systems that previously could not be completely interpreted by increases in the levels of cAMP(34) . The specific expression of G and G and the inability for -AR to couple to Gq, G, and G prevent the -AR ligands from activating PLC in most systems. This may serve as an example for how the signal transduction pathways are organized by both tissue-specific expression and selectivity in receptor-G protein interactions.

-AR appears to play important roles in the hematopoietic systems, and it provides a link between the neuroendocrine system and the immune system(35, 36, 37, 38) . The expression levels of -AR in leukocytes vary in some pathological conditions, such as asthma(39) , rheumatoid arthritis, and Crohn' s disease(40) . Most previous research was directed at cAMP, which is certainly one of the messengers downstream of the receptor. However, activation of PLC by -AR through G should also be investigated as a possible signal transduction pathway for -AR-mediated effects in hematopoietic systems. It would be interesting to understand why -AR couples to two signal transduction pathways in the hematopoietic system and what the precise physiological function of -AR-mediated activation of PLC is.


FOOTNOTES

*
This work was supported by the Pharmaceutical Research and Manufacturers of America Foundation, Inc. (to D. W.) and by the Leukemia Society of America (to H. J.). 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.

§
To whom correspondence should be addressed.

The abbreviations used are: PLC, phospholipase C; AR, adrenergic receptor; IP(s), inositol phosphate(s).

D. Wu, unpublished results.


ACKNOWLEDGEMENTS

We acknowledge Dr. Allen Smrcka and Mark Betz for reading the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.