©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different 1-Adrenergic Receptor Sequences Required for Activating Different G Subunits of Gq Class of G Proteins (*)

Dianqing Wu (§) , Huiping Jiang , Melvin I. Simon (1)

From the (1) Department of Pharmacology, University of Rochester, Rochester, New York 14642 Division of Biology, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In order to understand the specific interactions between receptors and guanine nucleotide-binding regulatory protein (G proteins), we attempted to delineate the 1B-adrenergic receptor sequences involved in activation of the subunits of the Gq class of G proteins. A number of specific mutations were introduced into the third inner loop of the receptor, and the mutants were tested for their abilities to activate different G subunits of the Gq class. Our results indicate that the receptor sequences required for activating Gq/11, G14, or G16 are different. The sequence extending from residues Lysto Hisis required for activation of Gq/11, but not for activation of G14 or G16. Two segments in the third loop of the receptor are required for activation of G14: one is located at the N terminus of the loop ending at residue Asn, and the other is located at the C terminus of the loop starting from residue Ser. The latter contains a BB XXB motif, which is apparently critical for G14 coupling, but not for G16 or Gq/11 coupling. Furthermore, the three amino acids stretch (Tyrto Val) included in the N-terminal segment is not only required for G14 coupling, but also for Gq/11 coupling. It may be involved to some extent in G16 coupling as well.


INTRODUCTION

Molecular cloning has revealed the existence of genes encoding at least 16 G subunits, 5 G subunits, and 7 G subunits in mammals. These can form a variety of heterotrimers that serve to connect specific cell surface receptors to a large number of different effectors, including at least four PLC() isoforms and many adenylylcyclases as well as several specific ion channels (1, 2, 3) . One of the intriguing questions posed by this apparent complexity is how signal transduction circuits are organized so that different kinds of receptors can be connected to effectors through various G proteins and coordinate a variety of responses in a large number of different cells. The specificity of some of the circuits is no doubt determined by developmental regulation of the expression of genes that encode the receptors, G proteins, and effectors. In addition, subcellular localization may contribute to the specificity to a certain extent. However, the primary determinant for formation of a specific signal transduction circuit, we believe, lies in specific protein-protein interactions.

In general, four methods have been used to detect the specific interactions between receptors and G proteins: 1) the use of specific antibodies to block ligand-induced activation of PLC in membrane preparations or to immunoprecipitate G proteins that are photolabeled with a GTP analog. A number of laboratories have used these approaches to detect specific coupling of receptors, including those for vasopressin (4) ,thromboxane A2 (5) , bradykinin, vasopressin, angiotensin (6) , and thyrotrophin-releasing hormone (7) , to Gq and/or G11. 2) The use of antisense oligonucleotides to inhibit expression of various subunits of G proteins. This approach has been successfully applied to reveal the specific interactions among the receptors, G, G, and G in GH3 cells (8, 9) . 3) The use of purified proteins in reconstituted systems (2, 10, 11, 12) . 4) The use of the cotransfection system, in which cells are transfected with cDNAs encoding receptors and G proteins with subsequent measurement of ligand-induced responses. This approach has been used to study coupling of the IL-8 (13) , C5a (14) and -adrenergic (15, 16, 17, 18) receptors to the subunits of the Gq and Gi classes. By using these methods, we and others found that there is clear specificity in receptor-G protein interactions. For instance, the 1-adrenergic receptors preferentially couple to the subunits of the Gq class of G protein to activate inositide-specific PLC, while the -adrenergic receptors couple to the Gproteins (19) . Furthermore, the specificity lies not only with different classes of G proteins, but also within the same class. For example, we found that the IL-8 receptor can couple to G16 and G14, but not to Gq or G11 (13) .

A lot of work has been done to understand the molecular basis of the specificity in receptor-G protein interactions (20) . Amino acid sequences that are involved in activation of Gq have been mapped to the third cytoplasmic (inner) loops of the 1B-adrenergic receptor, the m1 muscarinic receptor, and the glutamate receptors by using various chimeras (21, 22, 23) . Although these sequences share no significant amino acid sequence homology, they appear to be different from the sequences involved in activating Gs (24, 25) . It is, however, not known whether these Gq-activating sequences are also involved in activating the other members of the Gq class, such as G14, G15, and G16, and how receptors like the 1B-adrenergic receptor can couple to all G subunits of the Gq class, while receptors like the IL-8 receptor couple to G14, G15, and G16, but not to Gq/11. In this report, we investigated these questions by using site-directed mutagenesis on the third loop of the 1B-adrenergic receptor. Our results indicate that different 1B-adrenergic receptor sequences are involved in coupling to Gq/11, G14, or G16.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

COS-7 cells were cultured in Delbecco's modified Eagle's medium containing 10% fetal calf serum under 5% COat 37 °C. For transfection, COS-7 cells were seeded into 12-well plates at a density of 1 10cell/well the day before transfection. The media were removed the next day, and 0.5 ml of Opti-MEM (Life Technologies, Inc.) containing 5 µg of lipofectamine (Life Technologies, Inc.) and 1 µg of plasmid DNA was added to each well. Five h later the transfection media were replaced by the culture media. Then the cells were labeled with 10 µCi/ml of myo-[2-H]inositol the following day, and the levels of inositol phosphates were determined 1 day later as described previously (26) . All the cDNAs used in this studies were constructed in the pCMV expression vector (26) .

SDS-Polyacrylamide Gel Electrophoresis and Western Blot

Equal numbers of transfected cells were solubilized in the SDS sample buffer and loaded to 12% SDS-polyacrylamide gels. The proteins were then electroblotted to nitrocellulose membranes and detected with antibodies indicated in the figure legends.

Binding Assays

COS-7 cells in 12-well plates were transfected with the cDNA encoding the 1-adrenergic receptor or its mutants. After 48 h, the cells were washed with phosphate-buffered saline and incubated with varying amounts of [H]prazosin (76 Ci/mmol, PuPont-NEN) in phosphate-buffered saline for 40 min at 4 °C. After washing three times with ice-cold phosphate-buffered saline, the cells were lysed in 0.5 ml of 0.2 N NaOH, and 0.1-ml aliquots were taken for counting in a scintillation counter. The nonspecific binding was determined by measuring binding of [H]prazosin to nontransfected cells. The number of specific binding sites ( Bmax) were determined by the Scatchard analysis.

The half-maximal inhibition value (IC) was determined by measuring binding of [H]prazosin (5 pM) to transfected COS-7 cells in the presence of varying amounts of norepinephrine.

Construction of 1-Adrenergic Receptor Mutants

All the 1-adrenergic receptor mutants listed in Fig. 1 were generated by polymerase chain reaction with the high fidelity DNA polymerase, pfu (Stratagene) and each of the mutations was confirmed by DNA sequencing.


Figure 1: Schematic representation of 1B-adrenergic receptor sequences required for activation of subunits of Gq class. The amino acid sequence of the third inner loop of the 1B-adrenergic receptor is shown. The various deletion mutations are denoted and so is the residue Lys, which is substituted by a Leu residue in mutant 1BK282L. The ligand binding activities were determined as described under ``Experimental Procedures.''




RESULTS

We have previously demonstrated that the 1B-adrenergic receptor can couple to all the G subunits of the Gq class of G proteins, including Gq, G11, G14, and G16, to activate PLC (15) . As shown in Fig. 2 A, COS-7 cells transfected with the 1B-adrenergic receptor cDNA showed norepinephrine-dependent increases in accumulation of inositol phosphates (IP). This suggests that the 1B-adrenergic receptor can couple to endogenous G proteins, which are probably Gq and G11, because Gq and G11, but not G14, G15, or G16, were detected in COS-7 cells (15) . When the cells were cotransfected with the cDNA encoding G14 or G16, there were further increases in ligand-dependent responses over the ones seen in the cells transfected with the 1B-adrenergic receptor alone (Fig. 2 A), suggesting that the 1B-adrenergic receptor can couple to G14 and G16 as well. In order to determine whether the 1B-adrenergic receptor sequences involved in activating G14 and G16 are the same as the ones required for activating Gq/11 (22) , we made a specific mutation in the 1B-adrenergic receptor by deleting amino acids from residues Lysto His(Fig. 1). This 13-amino-acid segment was previously identified as involved in Gq/11 coupling (22) . As expected, the cells transfected with the cDNA encoding this mutated 1B-adrenergic receptor, designated 1B2, unlike those transfected with the wild-type receptor cDNA, showed little ligand-induced accumulation of IPs (Fig. 2, B and C, open circles). Even cotransfection with the cDNA encoding Gq or G11 led to little ligand-induced response (data not shown), confirming that this segment of the 1B-adrenergic receptor is required for coupling to Gq or G11. However, when the cells were cotransfected with the 1B2 cDNA and the cDNA encoding either G14 (Fig. 2 B) or G16 (Fig. 2 C), there were marked increases in norepinephrine-induced accumulation of IPs. The expression levels of the recombinant G14 and G16 proteins in transfected COS-7 cells were determined by the Western analysis with a polyclonal antibody (Gqcom), which was raised against a synthetic peptide derived from the sequence shared by all the G subunits of the Gq class (Fig. 3 A). The Western analysis indicated that G14 is expressed at a slightly higher level than some endogenous proteins detected by the Gqcom antibody in nontransfected COS-7 cells; these endogenous proteins are presumably Gq and G11. The identities of the endogenous proteins and expression of G14 were further confirmed by the Western analysis using the Gq/11- and G14-specific antibodies. G14 was detected only in cells transfected with the G14 cDNA by the G14-specific antibody (Fig. 3 A) and a clear band, which has the same electrophoretic mobility as the recombinant Gq, was detected by the Gq/11-specific antibody in nontransfected COS-7 cells (Fig. 3 B). In addition, we determined the expression levels of the wild-type 1B-adrenergic receptor and its mutant 1BAR and their affinities for norepinephrine. The expression levels were determined by binding of [H]prazosin to the cells expressing the receptors, while the binding affinities are indicated by the IC, which is the concentration of norepinephrine showing 50% inhibition of binding of [H]prazosin to the cells expressing the 1B-adrenergic receptor or its mutants. We found that the cells expressing the 1-adrenergic receptor or 1B2 showed similar numbers of binding sites for [H]prazosin (about 500 fmol/1 10cells) and similar ICvalues (7 µM) (Fig. 1). Furthermore, we found that coexpression of different G proteins does not affect expression of either 1B-adrenergic receptor or 1B2 in cotransfected COS-7 cells (data not shown). Therefore, we conclude that the sequence extending from residues Lysto Hisis required for activating Gq and G11, but not for activating the other G subunits of the Gq class, including G14 and G16, and that there might be different sequences involved in coupling to G14 or G16.


Figure 2: Activation of the Gq subunits by wild-type 1BAR and its mutants, 1B2 and 1B11. A, COS-7 cells were cotransfected with the cDNA encoding 1B-adrenergic receptor and the cDNA encoding G14 ( closed triangle), G16 ( closed circle), or the control -galactosidase ( open square). B and C, COS-7 cells were cotransfected with the cDNA encoding 1B2 ( circles) or 1B11 ( triangles), and the cDNA encoding G14 ( B, closed symbols), G16 ( C, closed symbols), or the control -galactosidase ( open symbols). The levels of inositol phosphates were determined 20 min after addition of ligand. The level of inositol phosphates in nontransfected COS-7 cells is taken as 100%.




Figure 3: Western analysis of expression of G subunits in COS-7 cells. Cells transfected with the cDNA encoding G14, G16 ( A), Gq ( B) or the control -galactosidase (LacZ) were solublized in the SDS sample buffer and separated by 12% SDS-polyacrylamide electrophoresis gels. The proteins were then electrotransferred to nitrocellulose membranes and analyzed by Western blotting with the Gqcom antibody ( A, left panel), G14-specific antibody ( A, right panel), or Gq/11-specific antibody ( B).



To delineate the 1B-adrenergic receptor sequences involved in activating G14 or G16, we generated the mutant 1B11 by deleting the amino acids from residues Argto Phein the 1B-adrenergic receptor. Although this mutant showed altered ligand-binding characteristic (lower IC) and was expressed at a lower level than the wild-type and 1B2 (Fig. 1), it can still activate G14 and G16 with capabilities comparable to the 1B2 (Fig. 2, B and C). This indicates that the 1B-adrenergic receptor amino acids extending from residues Leuto Pheare not required for coupling to G14 or G16.

One of the interesting questions is whether the sequences required for activating G16 can be separated from those for activating G14. To address this question, we made another mutant, 1B1, with a deletion extending from residues Alato Ser(Fig. 1). The mutant 1B1 was tested in the same way as for 1B2, and we found that 1B1 could couple only to G16, but not to G14 (Fig. 4 A). This tells us that the sequence required for activating G16 is different from the one required for activating G14. In addition, the fact that 1B1 can not couple to G14, while 1B11 can, suggests that the sequence extending from residues Alato Asnis required for activating G14. In order to further delineate the sequences required for activating G14, we prepared two additional mutants, 1B3 and 4 (Fig. 1). The inability of 1B4 (Fig. 4 C) and the ability of 1B3 to activate G14 (Fig. 4 B) indicate that the 1B-adrenergic receptor sequence extending from residues Serto Alais also required for activating G14. Thus, there appear to be two segments involved in G14 coupling: one is located near the C-terminal end and the other near the N-terminal end of the third inner loop of the 1B-adrenergic receptor. Although 1B4 could not activate G14, it as well as 1B3 could still activate G16. Activation of G16 thus acts as an internal control for 1B4, indicating it retains overall structural integrity. All the 1B mutants were tested for their expression levels and ligand binding affinities. The numbers of binding sites and the ICvalues shown by cells expressing the mutants 1, 3, and 4 are between those shown by 1B2 and 11 (Fig. 1).


Figure 4: Activation of the G subunits by 1-adrenergic receptor mutants. COS-7 cells were cotransfected with the cDNA encoding one of the 1BAR mutants as indicated in the figure and the cDNA encoding G14, G16, or the control -galactosidase (LacZ, LZ). The levels of inositol phosphates were determined 20 min after addition of norepinephrine (1 µM). The level of inositol phosphates in nontransfected COS-7 cells is taken as 100%.



Interestingly, the two segments required for activation of G14 contain a signature motif, BB XXB (B represents a basic amino acid and X represents any amino acid) (Fig. 1). This motif occurs frequently in the inner loops of many G protein-coupled receptors and has been implicated as a part of consensus sequences involved in coupling to Gi (27) . To test whether this motif is important for G14 coupling, we introduced a point mutation into each of the motifs. The mutant 1BK282L with substitution of residue Lysfor a Leu residue at the C-terminal end of the third inner loop can couple to the endogenous Gq/11 to the same extent as the wild-type 1B-adrenergic receptor (Fig. 5 A). To test whether this mutation has any effect on coupling to G14 or G16, we introduced another mutation into the mutant 1BK282L by deleting the amino acid sequence extending from residues Lysto His, which is also deleted in 1B2 (Fig. 1), so that we can test the mutated receptor without activating endogenous Gq/11. This new mutant, designated 1B2KL, couples to G16 as well as 1B2 does (Fig. 5 B), but it cannot couple to G14 (Fig. 5 C). This result, in addition to the fact that both 1BK282L and 1B2KL showed similar ligand-binding characteristics and both are expressed at similar levels to the wild-type receptor and to 1B2 (Fig. 1), allows us to conclude that the sequence motif (BB XXB) located at the C-terminal end of the third inner loop is required for coupling to G14. A similar sequence motif that is located at the N-terminal end of the third loop was also mutated by substituting residue Lysfor an Ala residue in both the wild-type and 1B2 (Fig. 1). However, this mutation had no effect on coupling to Gq/11, G14, or G16 (data not shown), suggesting that neither residue Lysnor the entire motif is required for G protein coupling.


Figure 5: Activation of G subunits by mutants 1BK282L and 2KL. A, cells were transfected with the cDNA encoding 1BK282L ( squares) or the 1B-adrenergic receptor ( triangles). B, cells were cotransfected with the cDNA encoding 2KL ( squares) or 1B2 ( triangles) and the cDNA encoding G16. C, cells were cotransfected with the cDNA encoding 2KL ( right two bars) or 1B2 ( left two bars) and the cDNA encoding G14 or -galactosidase (LacZ, LZ). The levels of inositol phosphates were determined 20 min after addition of norepinephrine. 1 µM of norepinephrine was added in panel C. The level of inositol phosphates in nontransfected COS-7 cells is taken as 100%.



In an attempt to determine the sequences that are directly involved in activation of G16, we made the mutant 1B5 by deleting the 1B-adrenergic receptor sequence from residues Tyrto His. We found that this mutant, when cotransfected with any of the G subunits of the Gq class, showed no ligand-induced activation of PLC (Fig. 4 D). This suggests that it cannot couple to any of the G subunits, although it can still bind ligand albeit with a decreased ICand lower expression level (Fig. 1). This result raised a possibility that the three amino acids extending from Tyrto Valmay be involved in coupling to G16 because the mutant 1B5, which lacks these amino acids, loses its ability to activate G16, while the mutant 1 that contains these three amino acids can still activate G16 (Fig. 4 A). To test this possibility, we generated two 1B-adrenergic receptor mutants: one, designated 1B12, was generated by deleting the three amino acids (Tyr-Val) in the wild-type 1B-adrenergic receptor and the other, designated 1B122, was generated by deleting these three amino acids in 1B2. The latter was made because it lacks the Gq/11-coupling sequence so that it can be tested for coupling to G14 and G16 in a background-free system. Both mutants were tested for their abilities to activate the G subunits of the Gq class. We found that COS-7 cells transfected with either of the mutants gave no ligand-dependent accumulation of IPs (Fig. 6), suggesting neither of the mutants could couple to the endogenous Gq/11. Moreover, these two mutants did not show ligand-dependent accumulation of IPs when they were coexpressed with G14 (Fig. 6). However, ligand-dependent accumulation of IPs was found when these two mutants were coexpressed with G16, but the responses were much lower than those found with the 1B2 mutant (Fig. 6). The binding assay indicates that 1B12 (Fig. 1) and 1B122 (data not shown) were expressed at lower levels (160 fmol compared to 500 fmol expressed by the wild-type) with no changes in their ICvalues, suggesting that the deletion somehow reduces the expression levels, without significantly changing the conformation of the ligand-binding domains. We do not think that reduction in the number of binding sites is sufficient to account for the lower responses found in cells coexpressing G16 because the mutant 1B11 that expresses at the similar level strongly activates G16. Thus, we conclude that though these three amino acids extending from residues Tyrto Valmay not constitute the major G16-interacting domain, they are involved in G16 activation and that these three amino acids are certainly required for activating Gq/11 and G14. Moreover, the same abilities of 1B12 and of 1B122 to activate G16 indicates that the 13 amino acids (Lys-His) deleted in the 2 mutants have no effect on specific G16 activation, thus suggesting that the 12-amino-acid sequence is essentially not involved in G16-coupling.


Figure 6: Activation of G subunits by mutants 1B12 and 1B212. COS-7 cells were cotransfected with the cDNA encoding 1B2, 1B12, or 1B212 and the cDNA encoding G14, G16, or -galactosidase (LacZ, LZ). The levels of inositol phosphates were determined 20 min after addition of norepinephrine (1 µM). The level of inositol phosphates in nontransfected COS-7 cells is taken as 100%.




DISCUSSION

In this report we used the site-directed mutagenesis and cotransfection approaches to analyze the third inner loop of the 1B-adrenergic receptor for its involvement in specific coupling to the subunits of the Gq class of G proteins and found that different 1B receptor sequences are required for coupling to Gq/11, G14, and G16. The amino acids extending from residues Lysto Hisand from residues Tyrto Valare required for activating Gq/11, while the amino acids upstream of residue Asnand downstream of residue Serin the third inner loop of the receptor are required for activating G14. It appears that the third inner loop of 1-adrenergic receptor does not play a crucial role in activation of G16.

The BB XXB motifs are commonly found in G protein-coupled receptors. There are two such motifs in the third inner loop of the 1B-adrenergic receptor. The second basic amino acid, Lysin the motif located at the C-terminal end of the third inner loop is required for activating G14. However, we do not know whether the whole motif at the C-terminal end of the third inner loop is part of the consensus sequences involved in G14 coupling. It is interesting to note that the C5a receptor (data not shown), which lacks this sequence motif at the C-terminal end of the third inner loop, cannot couple to G14, while the 1B-adrenergic receptor and IL-8 receptors, which have this motif at the corresponding positions, can couple to G14 (13) , suggesting that this motif may be part of the consensus sequence involved in G14 coupling. We will be able to test this hypothesis by studying more G14-coupling receptors.

Although our data focus on the third inner loop of the 1-adrenergic receptor, we do not exclude the possibility that other segments of the receptor may be involved in G protein binding and activation. In addition, we do not know if loss of coupling activities caused by specific mutations is a result of conformational changes or loss of specific interacting or activating peptide sequences. The results that all the mutated receptors retain ligand binding activities and that most of them showed ligand-dependent activation of G16 suggest that the modified receptors retain their overall structural integrity. In addition, we did not include G in our study. G has been shown to be involved in receptor-G protein coupling (12) . In this study, endogenous G subunits may be involved. However, since there appears to be little specificity in the interactions between G and G (28) or in G-mediated regulation of effectors (29, 30) , we believe that our results in this report would be largely unaffected by molecular differences in G.

The 1B-adrenergic receptor and some of its mutants apparently couple to G16 better than to G14. The fact that G14 is expressed at a lower level than G16 may partially explain this observation (Fig. 3 A). It is also possible that the 1B-adrenergic receptor has a lower affinity for G14 or/and that G14 is a less effective activator of PLC. Since it is difficult to use the cotransfection assay system for kinetic or quantitative studies due to the difficulties in manipulating the expression levels of recombinant proteins, we were unable to determine the precise mechanism. Moreover, it is worth noting that no differences between Gq and G11 have been observed in coupling to any of the mutated receptors (data not shown).

Unfortunately, in this study we were unable to delineate the sequences involved in activating G16. However, our results presented in this report indicate that the 1-adrenergic receptor sequences that determine the specificity in G16 coupling are unlikely to be located in the third inner loop of the receptor. A number of reports have previously indicated that the other inner loops and C-terminal end of G protein-coupled receptors also play roles in G protein coupling (31, 32) . Currently, we are investigating the other inner loops of the 1-adrenergic receptor for their roles in coupling to G16 by using similar approaches.

In summary, our work has provided useful information for understanding of specific interactions between receptors and G proteins. The data suggest that we may be able to create receptors with limited, but defined G protein-coupling specificity. Such receptors would be very useful in determining the specific in vivo function of signal transduction pathways mediated by specific receptors and G proteins.


FOOTNOTES

*
This work is supported by a National Institutes of Health grant (to M. I. S.) and a Leukemia Society of America fellowship (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: Dept. of Pharmacology, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-2029; Fax: 716-244-9283.

The abbreviations used are: PLC, phospholipase C; G protein, guanine nucleotide-binding regulatory protein; IL-8, interleukin 8; IP, inositol phosphates.


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