©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specificity of G Protein - Subunit Interactions
N-TERMINAL 15 AMINO ACIDS OF SUBUNIT SPECIFIES INTERACTION WITH alpha SUBUNIT (*)

(Received for publication, August 2, 1994; and in revised form, November 2, 1994)

Mohammed Rahmatullah Roman Ginnan Janet D. Robishaw (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The existence of multiple alpha, beta, and subunits raises questions regarding the assembly of particular G proteins. Based on the results of a previous study (Rahmatullah, M., and Robishaw, J. D.(1994) J. Biol. Chem. 269, 3574-3580), we hypothesized that the assembly of G proteins may be determined by the interactions of the more structurally diverse alpha and subunits. This hypothesis was confirmed in the present study by showing striking differences in the abilities of the (1) and (2) subunits to interact with the the alpha(o) subunit. Chimeras of the (1) and (2) subunits were used to delineate which region is responsible. Support for the importance of the N-terminal region of the subunit comes from our observations that 1) the (2) subunit and the chimera bound strongly to the alpha(o)-agarose matrix, but the (1) subunit and the chimera bound weakly, if at all; 2) an N-terminal peptide made to the (2) subunit blocked the binding of the chimera to the alpha(o)-agarose matrix; 3) both the chimera and the N-terminal peptide were able to partially protect the alpha(o) subunit against tryptic cleavage; and 4) the chimera, but not the chimera, supported ADP-ribosylation of the alpha(o) subunit.


INTRODUCTION

The ability of cells to process a large number of extracellular signals into the appropriate array of intracellular responses depends on the types of signaling pathways that are expressed in the plasma membranes of these cells. The most common type of signaling pathway relies on the interaction of three kinds of proteins: receptors, heterotrimeric G proteins, (^1)and effectors. The recent identification of more than 100 different types of receptors, more than 320 possible combinations of G protein heterotrimers, and an unknown number of effectors (1, 2) raises important questions as to how these signaling pathways are assembled to ensure the correct transmission of a signal from a particular receptor to a specific effector. Given its position between the receptor and the effector, it seems likely that much of the fidelity of signal transmission must reside in the alphabeta subunit structure of the G protein.

While it has generally been assumed that a G protein should be defined on the basis of the alpha subunit, the rapidly growing number of different beta and subunits suggests that a G protein should more properly be defined on the basis of its unique combination of alphabeta subunits. This idea is supported by antisense ``knockout'' studies by Kleuss et al.(3, 4, 5) , demonstrating different combinations of G protein alphabeta subunits couple somatostatin and muscarinic receptors to inhibition of the same Ca channel. However, since the alpha and the beta subunits undergo an appreciable rate of dissociation in the course of detergent extraction and purification, it has not been possible to confirm the alphabeta composition of a particular G protein by biochemical studies. Furthermore, the tight association of the beta subunits has not permitted the regions of the individual beta and subunits that interact with the alpha subunits to be examined.

In a previous paper(6) , we expressed the individual beta and subunits in the baculovirus system and showed the direct association of the (2) subunit with the alpha subunit of G(o) (alpha(o) subunit). This result suggested the possibility that selective association of the and alpha subunits might provide the mechanistic basis for the assembly of particular G protein alphabeta subunit combinations. With the recent identification of at least 10 different subunits (^2)(7) and with their expression in the baculovirus system, it is now possible to examine the specificity of alpha and subunit interactions in a systematic fashion. In the present paper, we show differential association of the (1) and (2) subunits with the alpha(o) subunit. Furthermore, we identify the N-terminal 15 amino acids of the subunit as the region responsible for conferring the specificity of association with the alpha subunit, using a combination of peptide and chimeric approaches.


EXPERIMENTAL PROCEDURES

Cloning of Recombinant Baculoviruses for the (1), , , (2), beta(1)(3), and beta(1)(5)Subunits for Expression in Sf9 Cells

The coding regions of the beta(1), (1), (2), (3), and (5) cDNAs were subcloned into the pVL1392 or pVL1393 transfer vectors as previously described(8, 9, 10) . Using a combination of restriction enzyme digestion and polymerase chain reaction, the chimeric cDNA was generated by replacing the C-terminal 23 amino acid residues of the (1) subunit with the corresponding 23 amino acid residues of the (2) subunit, while the chimeric cDNA was produced by replacing the N-terminal 18 amino acid residues of the (1) subunit with the corresponding 15 amino acid residues of the (2) subunit. Following polymerase chain reaction amplification, the chimeric and cDNAs were subcloned into the pVL1392 transfer vector. Transfer of the cDNAs from this transfer vector to the wild type Autographa californica nuclear polyhedrosis virus genome and plaque purification of the resulting recombinant viruses were performed as previously described(8, 9, 10) . The recombinant viruses were used to infect Spodoptera frugiperda (Sf9) cells at a multiplicity of infection of 2. The Sf9 cells were grown in monolayers or spinners in TNM-FH medium containing 10% fetal calf serum at 27 °C.

Purification of G Protein Subunits

The alpha(o) subunit was purified from bovine brain as previously described by Sternweis and Robishaw(11) . The alpha(s) subunit was obtained as a side product of the alpha(o) purification. Elution fractions from the C-7 column enriched in the alpha(s) subunit were pooled, concentrated on an Amicon ultrafiltration device with a PM-10 membrane, and then dialyzed for 16 h with 2 changes against buffer A (20 mM Tris-Cl (pH 8), 0.1 M NaCl, 6 mM MgCl(2), 1 mM EDTA, 10 mM sodium, 10 µM AlCl(3), 1 mM DTT, and 0.3% cholate). Further purification of the alpha(s) subunit on octyl-Sepharose was performed as previously described(12) . The beta(1)(3) and beta(1)(5) dimers were purified from the particulate fractions of baculovirus-infected Sf9 cells using alpha(o) affinity and hydroxyapatite chromatography as previously described(9, 10) . Elution fractions containing the beta(1)(3) and beta(1)(5) dimers were pooled and concentrated. Prior to use in ADP-ribosylation assays, the beta(1)(3) and beta(1)(5) dimers were dialyzed against two changes of buffer B (20 mM Tris-Cl (pH 8), 1.6 mM DTT, 1 mM EDTA, and 0.05% LPX).

Preparation of Cytosolic and Membrane Fractions from Sf9 Cells Infected with Subunits

Approximately 68 h after infection, the cells were collected from 1-liter spinner flasks by centrifugation (3,000 times g for 10 min), rinsed with phosphate-buffered saline, and resuspended in 40 ml of buffer C (20 mM Na-HEPES (pH 8), 2 mM MgCl(2), 1 mM EDTA, 2 mM DTT, 0.1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, and 1 mM benzamidine). The cells were lysed by Parr bombing at 500 p.s.i. for 45 min at 4 °C and then stirred gently on an orbital shaker for 3 h at the same temperature. The supernatants were collected by centrifugation (100,000 times g for 30 min) and designated as the cytoplasmic fraction. The pellets were dissolved in 40 ml of buffer C containing 1% cholate for 16 h at 4 °C by stirring on an orbital shaker. Any insoluble material was discarded by centrifugation at 100,000 times g for 30 min. The supernatant from the last centrifugation was designated as the particulate fraction.

Binding and AMF-specific Elution of Subunits from the alpha(o)-affinity Resin

To prepare the alpha(o)-affinity resin, approximately 30 mg of the purified alpha(o) subunit was coupled to 15 ml of -aminobutyl-agarose as described by Mumby et al.(13) . The resin was washed extensively with buffer D (20 mM Na-HEPES (pH 8), 400 mM NaCl, 1 mM EDTA, 2 mM DTT, and 5 µM GDP) containing 0.5% LPX prior to packing into a Pharmacia XK 16/20 column. To measure binding to the alpha(o)-affinity resin, we loaded equivalent amounts of the expressed subunits based on immunoblotting of cytosolic and particulate fractions with the same antibody, immunoblotting conditions, and exposures for detection. Accordingly, a 3-ml aliquot of the cytosolic and particulate fractions from - and (1)-infected cells or a 1-ml aliquot from -infected cells was diluted 10-fold with buffer D containing 0.05% LPX and then loaded onto the alpha(o)-affinity column at a flow rate of 0.3 ml/min. The column was then washed successively with 2 column volumes of buffer D containing 0.5% LPX at a flow rate of 0.9 ml/min and 20 column volumes of buffer D containing 0.05% LPX at a flow rate of 1.8 ml/min. The column was then eluted with buffer E (20 mM Na-HEPES (pH 8), 400 mM NaCl, 2 mM DTT, 20 mM MgCl(2), 10 mM sodium, 5 µM GDP, and 30 µM AlCl(3)) at a flow rate of 0.3 ml/min. The AMF elution fractions containing the subunits were pooled, concentrated, and then measured for protein concentration using the Amido Black assay.

Peptide Studies

To evaluate the importance of the N-terminal region of the subunit, a series of peptides made to the various regions of the (1) and (2) subunits were synthesized. To examine the effect of these peptides on (2) or binding, the alpha(o)-agarose matrix was pre-equilibrated with about 1 µmol of each of these peptides. This amount of each of the peptides was estimated to be 100-fold higher than the amount of (2) or loaded on the matrix, taking into account the possibility that the shorter sequence of the peptides might not be optimal toward adopting full conformation for binding to the alpha(o) subunit.

Tryptic Proteolysis Assay

Tryptic proteolysis assays were conducted with the alpha(o) or alpha(s) subunits in the presence or absence of the purified chimeras. Unless indicated otherwise, to 20 µl of assay buffer was added 10 µl of alpha(o) or alpha(s) subunits and up to 48 µl of the chimeric subunits (an equivalent amount of elution buffer concentrate from a mock alpha(o)-affinity column run was used as a control). The volume was brought up to 90 µl with water, and the mixture was incubated at 4 °C for 30 min followed by room temperature incubation for 10 min. Proteolysis was initiated by addition of 10 µl of trypsin at a constant protein to trypsin ratio of 50:1 and incubation at 30 °C. The final assay mix contained 35 mM Na-HEPES (pH 8), 300 mM NaCl, 2 mM DTT, 10 mM MgCl(2), 40 mM EDTA, 5 µM GDP, and 0.1% LPX. We found that this high concentration of EDTA was necessary due to the presence of AMF in the fractions utilized in these studies. 20-µl aliquots were withdrawn at timed intervals, and reactions were terminated by adding 15 µl of 2.3 times Laemmli sample buffer (containing 23 mM benzamidine) and boiling for 5 min at 90 °C.

ADP-ribosylation of alpha(o)by Pertussis Toxin

The procedure used was a modification of that described by Casey et al.(14) . To 10 µl of stock assay buffer was added 5 µl of purified alpha(o) and up to 20 µl of beta dimers or 10 µl of chimeric subunits. An appropriate volume of beta dialysis buffer or elution buffer was added to all samples to give equivalent concentrations of all reagents. The final concentrations of all reagents were 60 mM Tris-Cl (pH 8.0), 0.8 mM EDTA, 1 mM DTT, 5 mM MgCl(2), 80 µM GDP, 80 µM NaCl, 0.04% LPX, 2 mM NaF, 6 µM AlCl(3), 2 µM NAD, [P]NAD (12,000-15,000 cpm/pmol), 0.4 mM dimyristoylphosphatidylcholine, and 4 µg/ml pertussis toxin. Reactions were initiated by addition of [P]NAD and pertussis toxin and were allowed to proceed at 30 °C. Aliquots of 20 µl were withdrawn at 20 and 40 min of incubation, added to 20 µl of Laemmli 2 times sample buffer, and boiled for 5 min. Following SDS-polyacrylamide gel electrophoresis on 15% gels, ADP-ribosylation was visualized by autoradiography of dried gels.

Gel Electrophoresis and Immunoblotting

To follow the purification of the chimeric subunits, the proteins in the various chromatographic fractions were resolved by SDS-polyacrylamide gel electrophoresis on 15% acrylamide gels and transferred to nitrocellulose for immunoblotting as previously described(15, 16) . For immunoblotting, the antibody A-4 was used at a 1:500 dilution to detect (1), , and . The antibodies A-10, 584, U-49, B-17, B-53, and D-10 were used at a 1:500 dilution to detect the alpha(o), alpha(s), beta(1), (2), (3), and (5) subunits, respectively, as previously described (16, 17, 18) . The alpha(o), beta, and antibodies were detected with I-labeled goat anti-rabbit F(ab`)(2) fragment (DuPont NEN) (1 times 10^6 cpm/ml). The alpha(s) antibody was detected using horseradish peroxidase-conjugated anti-rabbit IgG chemiluminescent probe (Amersham Corp.).


RESULTS

Previously, we reported that the (2) subunit can stably interact with the alpha(o) subunit of G proteins(6) . This interaction was markedly enhanced by prenylation of the (2) subunit but was not dependent on the presence of the beta subunit. This result raised the possibility that selective interaction of particular alpha and subunits may provide the mechanistic basis for the assembly of specific alphabeta combinations of the G proteins. This possibility was confirmed in the present paper by showing marked differences in the abilities of the (1) and (2) subunits, which are only 36% identical at the amino acid level(19) , to interact with the alpha(o) subunit (Fig. 1).


Figure 1: Elution profile of the (1) and (2) subunits from the alpha(o)-agarose column. A portion of the 1% cholate extract (LYS) from Sf-9 cells infected with either (1) or (2) was loaded onto the alpha(o)-agarose column. The flow through (F) was collected. Following successive washes with buffer D + 0.5% LPX (high detergent wash, lane3) and buffer D + 0.05% LPX (lanes4-7), the G(o)alpha-agarose column was eluted with buffer E + 0.05% LPX containing AMF (lanes 8-18). The elution fractions (AMF) represent a pool of every three fractions after the addition of AMF (lanes 8-11) and thereafter a pool of every five fractions.



Use of a Chimeric Strategy to Identify the Region of the Subunit Conferring Selective Interaction with the alpha Subunit

To try to delineate the region(s) of the subunit responsible for determining the selective interaction with the alpha(o) subunit, chimeras were constructed to look for ``gain of function'' by replacing regions of the (1) subunit with the (2) subunit. For the chimera, the C-terminal 23 amino acid residues of (1) were replaced with the corresponding 23 residues of the (2) subunit (Fig. 2). This region was chosen since previous papers (6, 9) showed that prenylation of the subunit was shown to be necessary for stable interaction with the alpha subunit, suggesting a potential involvement of the C-terminal region of the subunit in alpha- interaction. For the chimera, the N-terminal 18 amino acid residues of (1) were replaced with the corresponding 15 residues of the (2) subunit. This region was selected since it was the most diverse at the amino acid level, suggesting the N-terminal region of the subunit as a possible site of interaction with the structurally diverse alpha subunits(19) . To direct the expression of the chimeric and wild type subunits, Sf9 cells were infected with recombinant baculoviruses encoding either the (1), , or subunits. Approximately 68 h after infection, the cytosolic and particulate fractions were prepared from these cells for analysis. The cytosolic and particulate fractions were analyzed separately since prenylated forms of the (1), , and subunits have been found in both fractions. (^3)It was previously shown that only the prenylated forms of the subunits have the potential to interact with the alpha subunit(6) . To monitor alpha- association, the chimeric and wild type subunits from either the cytosolic or particulate fraction were loaded onto an alpha(o)-agarose column under conditions favoring subunit association (i.e. in the presence of GDP)(6) . After extensive washing, the subunits were selectively eluted from the alpha(o)-agarose column under conditions favoring subunit dissociation (i.e. in the presence of AMF). To follow their binding and selective elution, an antibody raised to a peptide common to the (1), , and subunits was used (depicted by the stippledareas in Fig. 2). A consequence of utilizing the same antibody to detect both the chimeric and wild type proteins was that roughly equivalent amounts of the (1), , and subunits could be loaded, allowing their relative affinities for the alpha(o) subunit to be differentiated.


Figure 2: Construction of chimeric subunits. The regions of the (1) and (2) subunits that were switched to generate the chimeric and subunits are shown. To monitor the expression and purification of the (1), , and subunits, an antibody raised to a peptide common to these subunits was used, as shown by the stippledareas.



The elution profiles of the cytosolic and particulate fractions from (1)-infected cells are shown in Fig. 3. Both the cytosolic and particulate forms of the (1) subunit were detected in the flow through and 0.5% Lubrol wash fractions (lanes2 and 3) but not in the subsequent 0.05% Lubrol wash fractions (lanes4-7) or AMF elution fractions (8-18) (panelsA and B, topinsert). The elution profiles of the cytosolic and particulate fractions from -infected cells were similar. Using the same immunoblotting conditions that were used for the detection of the (1) subunit, both the cytosolic and particulate forms of the chimera were detected in the flow through and 0.5% Lubrol wash fractions (lanes2 and 3) but not in the AMF elution fractions (lanes8-18) (panelsA and B, middleinserts). However, after prolonged exposure of the autoradiogram for 5 days, we observed a low but detectable amount of the particulate form of the chimera in the AMF elution fractions (data not shown). The presence of the chimera was further confirmed by concentration of the AMF elution fractions followed by detection with antisera. The same procedure failed to detect either the cytosolic or particulate form of the (1) subunit in the AMF elution fractions (data not shown). Taken together, these results suggest that the (1) subunit shows no detectable binding to the alpha(o)-agarose matrix, while the chimera shows very low binding.


Figure 3: Elution profile of the chimeric subunits from the alpha(o)-agarose column. A portion of the 1% cholate extract (LYS) from cytoplasmic (panelA) or membrane (panelB) fractions from Sf-9 cells infected with (1) (top), (middle), or (bottom) was loaded onto the alpha(o)-agarose column. The flow through (F) was collected. Following successive washes with buffer D + 0.5% LPX (high detergent wash, lane3) and buffer D + 0.05% LPX (lanes 4-7), the G(o)alpha-agarose column was eluted with buffer E + 0.05% LPX containing AMF (lanes 8-18). The elution fractions (AMF) represent a pool of every three fractions after the addition of AMF (lanes 8-11) and thereafter a pool of every five fractions.



In contrast, the elution profiles of the cytosolic and particulate fractions from chimera-infected cells were markedly different (Fig. 3). Using the same immunoblotting conditions that were used for the detection of the (1) and subunits, a significant portion of the cytosolic form of the chimera was stably retained on the alpha(o)-agarose matrix and specifically eluted with buffer containing AMF (panelA, bottominsert). Interestingly, elution with AMF appeared to result in the resolution of the cytosolic form of the chimera into two peaks (lanes8-9 and 13-16, respectively). Although the reason for this is not known, the two peaks may be related to the nature of the prenyl group. In support of this possibility, we showed that the cytosolic form of the (1) subunit is modified by both farnesyl and geranylgeranyl moieties when expressed in Sf9 cells.^3 Since the chimera contains the prenylation recognition sequence of the (1) subunit, it is likely that the chimera is similarly modified by both farnesyl and geranylgeranyl groups. Thus, the two peaks may represent forms of the chimera with different prenyl moieties. Likewise, a significant portion of the particulate form of the chimera was also selectively retained and specifically eluted with AMF (panelB, bottominsert). However, unlike the cytosolic form, the particulate form of the chimera was eluted in a single peak (lanes8-10). Again, this may be related to the nature of the prenyl group and/or the extent of carboxyl methylation.^3 Nevertheless, it is clear from these results that replacement of the N-terminal 18 amino acids of the (1) subunit with the corresponding 15 amino acids of the (2) subunit is sufficient to confer the specificity of interaction with the alpha(o)-agarose matrix.

Ability of N-terminal (2)Peptide to Block the Interaction of the Chimera with the alpha(o)-agarose Matrix

To further examine the role of the N-terminal region of the subunit in specifying interaction with the alpha subunit, two peptides corresponding to different regions of the (2) subunit were synthesized. Peptide 1 contained the sequence ASNNTASIAQARK corresponding to amino acid residues 2-14 of the (2) subunit, while peptide 2 contained the sequence DLMAYSEAHAK corresponding to amino acid residues 35-46 of the (2) subunit(19) . Following equilibration of the alpha(0)-agarose matrix with each peptide, the selective retention and AMF-dependent elution of the chimera was monitored. As shown in Fig. 4, in the presence of peptide 1, the proportion of chimera that was bound and eluted with AMF was drastically reduced (panelB). In contrast, peptide 2 corresponding to the middle region of the (2) subunit resulted in little apparent reduction in the proportion of the chimera that was bound and selectively eluted from the column, although the pattern of elution was altered somewhat (compare panelsC and A). Peptides 1 and 2 had similiar effects on the binding of the (2) subunit (data not shown). Taken together, the marked difference in the abilities of the peptides 1 and 2 to inhibit the interaction of the chimera and (2) subunit with the alpha(o)-agarose matrix underscores the importance of the N-terminal region of the subunit for interaction with the alpha subunit. Interestingly, the middle region of the subunit has been implicated in the interaction with the beta subunit (20) .


Figure 4: Elution profile of the subunit from the alpha(o)-agarose column in the presence of N-terminal or mid-region (2) peptides. The alpha(o)-agarose column was loaded with 8 ml of a 0.15 mM solution (in buffer D + 0.05% LPX) of peptide 1 (panelB) or peptide 2 (panelC) and allowed to sit for 1.5 h prior to loading with the cytoplasmic form of the chimera. PanelA shows retention and elution of the subunit in the absence of peptides, while panelsB and C show retention and elution of the subunit in the presence of the N-terminal and internal (2) peptides, respectively.



Varied Ability of the and Chimeras to Protect alpha(o)and alpha(s)against Tryptic Cleavage

Previously, we showed that the (2) subunit, in the absence of the beta subunit, could protect the alpha(o) subunit against tryptic cleavage(6) . Accordingly, we examined whether the and chimeras could protect the alpha(o) subunit from tryptic cleavage. Because of the presence of AMF in the purified preparations of the and chimeras, tryptic digestions were carried out in the presence of a high concentration of EDTA. As shown in Fig. 5, tryptic cleavage of the 39-kDa alpha(o) subunit (lane1) generated a 37-kDa fragment (panelA, lane2) in the absence of any subunit. Interestingly, the addition of the chimera was able to partially protect the alpha(o) subunit against tryptic cleavage with increasing protection observed as the to alpha(o) ratio was raised from 0.25:1 up to 2:1 (lanes3-6). On the other hand, the addition of the chimera offered little or no protection even at the highest molar ratio of 2:1 (compare lane6 of each). These results confirm the importance of the N-terminal region of the subunit in promoting alpha- interaction. However, it should be noted that the chimera was less effective at protecting the alpha(o) subunit than the wild type (2) subunit. At a stoichiometry of 2:1, the wild type (2) subunit was found to fully protect the alpha(o) subunit against tryptic proteolysis(6) . Whether this difference is indicative of other regions of the subunit being involved in the protection of the alpha(o) subunit against trypsin or a reflection of lower affinity for the alpha(o) subunit because of the chimeric nature of remains to be established.


Figure 5: Effect of varying molar ratios of or on tryptic cleavage of G(o)alpha and G(s)alpha. Purified G(o)alpha (panelA) or G(s)alpha (panelB) was mixed with varying concentrations of the affinity-purified or subunit for 30 min at 4 °C and 10 min at room temperature prior to addition of trypsin at a constant protein:trypsin ratio of 50:1. Digestions were carried out for 60 min at 30 °C. The stoichiometries of alpha(o): or alpha(s): were 1:0.25 (lane3), 1:0.5 (lane4), 1:1 (lane5), and 1:2 (lane6). Lanes1 and 2 represent alpha(o) or alpha(s) in the absence or presence of trypsin, respectively, in the absence of any subunit.



We also decided to examine whether the addition of the chimera was equally able to protect the alpha subunit of G(s) (alpha(s) subunit) against tryptic cleavage. As shown in panelB of Fig. 5, the alpha(s) subunit (lane1) was digested rapidly by trypsin (lane2). Moreover, even at the highest stoichiometry of to alpha(s) of 2:1 (lane6), the chimera showed no protection of the alpha(s) subunit against tryptic proteolysis. Taken together, these results eliminate the possibility of the subunit acting as an inhibitor of trypsin, and as such, protecting the alpha subunit. More importantly, these results suggest that structural differences in the alpha subunits as well as the subunits are critical in specifying their interactions.

Ability of a Peptide Made to the N-terminal Region of (2)to Partially Protect alpha(o)against Tryptic Proteolysis

Since peptide 1 corresponding to amino acid residues 2-14 of the (2) subunit was able to inhibit the binding of the chimera to the alpha(o)-agarose matrix (Fig. 4A), we decided to evaluate the possible effect of this peptide to protect the alpha(o) subunit against tryptic proteolysis. By way of comparison, peptide 3 corresponding to amino acid residues 2-15 of the (1) subunit was also evaluated. As shown in Fig. 6, the alpha(o) subunit (lane1) was digested to 37- and 24- kDa fragments (lane2). Increasing concentrations of peptide 1 resulted in greater protection of the 39-kDa band and correspondingly less generation of the 37- and 24-kDa fragments (lanes3 and 4). In contrast, peptide 3 showed no protection of the 39-kDa band over the same concentration range (lanes5 and 6). Taken together, these results demonstrate a specific interaction between peptide 1 and the alpha(o) subunit. However, it should be noted that a 24:1 stoichiometry of peptide 1 to alpha(o) afforded less protection against tryptic proteolysis than a 2:1 stoichiometry of chimera to alpha(o). The higher stoichiometry required for peptide 1 compared with chimera may reflect the involvement of other regions of the subunit in interaction with the alpha subunit. Alternatively, other regions of the subunit may reduce the number of possible conformations, thereby increasing the proportion of the subunit in the correct conformation for interaction with alpha subunit.


Figure 6: Effect of N-terminal (2) and (1) peptides on tryptic cleavage of G(o)alpha. Purified G(o)alpha (3.3 µM) was incubated with peptide 1 (N-terminal (2) peptide) or peptide 3 (N-terminal (1) peptide) for 1.5 h at 4 °C and 10 min at room temperature prior to addition of trypsin at a constant alpha(o):trypsin ratio of 50:1. Digestions were carried out for 60 min at 30 °C. From left to right: alpha(o) in absence of trypsin (lane1) and presence of trypsin (lane2); trypsin digestion of alpha(o) in presence of 13.4 µM (lane3) and 84 µM (lane4) peptide 1; and trypsin digestion of alpha(o) in presence of 13.4 µM (lane5) and 84 µM (lane6) of peptide 3.



Varied Ability of and Chimeras to Support Pertussis Toxin-dependent ADP-ribosylation of alpha(o)

The beta dimers have been shown to support the pertussis toxin-dependent ADP-ribosylation of the alpha(o) subunit(14) . Accordingly, we decided to evaluate the ability of the and chimeras to promote ADP-ribosylation of the alpha(o) subunit. Somewhat to our surprise, the chimera was able to support ADP-ribosylation of the alpha(o) subunit (lanes8-11) though not to the extent observed with either the beta(1)(3) dimer (lanes2-4) or the beta(1)(5) dimer (lanes 5-7) at 15-fold lower concentrations (Fig. 7). Nevertheless, the ability of the chimera to support ADP-ribosylation of the alpha(o) subunit appeared to be real since increasing concentrations of resulted in increasing ADP-ribosylation of the alpha(o) subunit (lanes 8-11versus control in lane1). On the other hand, the chimera failed to support ADP-ribosylation of the alpha(o) subunit over the same concentration range (lanes 12-15).


Figure 7: Support of ADP-ribosylation of the alpha(o) subunit by beta and subunits. Purified G(o)alpha (0.55 µg) was ADP-ribosylated in the presence of beta(1)(3), beta(1)(5), , or subunits for 20 min at varying molar ratios of alpha(o) to subunits. Assay conditions were described under ``Experimental Procedures.'' From left to right: alpha(o) alone (lane 1) and in the presence of beta(1)(5) (lanes 2-4) or beta(1)(3) (lanes 5-7) at molar ratios of 0.2, 0.5, and 1, respectively, to alpha(o) or in the presence of (lanes 8-11) or (lanes 12-15) at molar ratios of 0.3, 0.9, 1.8, and 3, respectively, to alpha(o).




DISCUSSION

The existence of multiple alpha, beta, and subunits, many of which are expressed in the same tissue, raises questions regarding the assembly of G proteins with distinct roles in receptor-effector coupling. Hypothetically, the assembly of particular combinations of alpha, beta, and subunits might be regulated by differential expression of these subunits in various cell types and/or differential affinities of these subunits to associate with each other. If we extrapolate from previous findings on beta- (21, 22) and alpha- (6) interactions, it seems likely that the assembly of particular combinations of alpha, beta, and subunits may be determined by their differential affinities. It further seems plausible that functional diversity of the G proteins may be conferred by the specific assembly of the more structurally diverse alpha and subunits rather than the less structurally diverse beta subunits. This hypothesis was confirmed in the present study by showing striking differences in the abilities of the structurally diverse (1) and (2) subunits to interact with the alpha(o) subunit.

The N-terminal Region of the Subunit Specifies alpha- Interaction

In the present study, we have used chimeras of the (1) and (2) subunits to identify which region specifies interaction with the alpha subunit. Support for the importance of the N-terminal region of the subunit comes from our observations that 1) the (2) subunit and the chimera bound strongly to the alpha(o)-agarose matrix, but the (1) subunit and the chimera bound only weakly, if at all; 2) a, N-terminal peptide corresponding to amino acids 2-14 of the (2) subunit blocked the binding of the (2) subunit and the chimera to the alpha(o)-agarose matrix, but an internal peptide corresponding to amino acids 35-46 of the (2) subunit had little effect on binding; 3) the chimera and the N-terminal peptide were able to partially protect the alpha(o) subunit against tryptic cleavage; and 4) the chimera was able to partially support the ADP-ribosylation of the alpha(o) subunit, but the chimera had no such effect. Taken together, these results strongly indicate that the N-terminal region of the subunit plays the determinant role in conferring the specificity of interaction with a particular alpha subunit.

In an earlier report(6) , we showed that prenylation of the subunit is important for alpha- interaction. The present results extend that observation. Following expression in Sf9 insect cells, the (2) subunit is modified exclusively by a geranylgeranyl moiety, whereas the (1) subunit is modified by farnesyl and geranylgeranyl moieties in roughly equal proportions.^3 Nevertheless, even though a significant proportion of the (1) subunit is modified by a geranylgeranyl group, the (1) subunit cannot interact with alpha(o) subunit within the limits of detection. Thus, the N-terminal region of the subunit rather than prenylation of the C-terminal region plays the primary role in determining the specificity of the alpha- interaction. However, it is possible that the nature of prenyl group may play a secondary role in determining the specificity and/or stability of the alpha- interaction. Nevertheless, it is clear from these studies (6) that both a favorable N-terminal region and a prenylated C-terminal region of the subunit are needed for optimal alpha- association. Interestingly, a recent study by Neer and Spring (20) has implicated the middle region of the subunit in determining the specificity of beta- interaction. Taken together, these results would predict a model in which the N- and C-terminal regions of the subunit come together to form the face for alpha interaction while the middle region forms the opposing face for beta interaction.

The results of the present study support the computer-based prediction of the N-terminal regions of both alpha and subunits as their possible sites of interaction. In this regard, a computer-based analysis of coiled coil forming probabilities has led to the hypothesis that G protein heterotrimers form via triple-stranded coiled coil interactions involving the N-terminal regions of the alpha, beta, and subunits(23) . However, this is the first experimental evidence to support the involvement of the N-terminal region of the subunit in this interaction.

Role of the beta Subunit in Heterotrimer Formation

In a previous study(6) , we observed that the beta subunit, when expressed by itself, cannot bind to the alpha(o)-agarose matrix. Thus, the subunit appears to be necessary for conferring this binding. On the other hand, the highest coiled coil forming probabilities are present in the N-terminal domains of the beta subunits, with the N-terminal domains of the alpha and subunits being more variable(23) . Certainly, the formation of a double-stranded coiled coil between the beta and subunits could be further stabilized on formation of a triple-stranded coiled coil with the alpha subunit. Since the beta subunits exist as a dimer in the physiological state, one possible role for the beta subunit may be to confer additional stability to the heterotrimer. Another possible role for the beta subunit may be to allow conformational flexibility within the beta dimer enabling further interaction with the alpha subunit. For instance, the (1) subunit cannot bind to the alpha(o) subunit. However, the (1) subunit in association with the beta(1) subunit can bind to the alpha(o) subunit(9) . In future studies, we will express the chimeric subunits with the various beta subunits and study their combined interactions with the alpha subunit. It is expected that such results will eventually provide the mechanistic basis for G protein heterotrimer formation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM39867 (to J. D. R.). 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: Weis Center for Research, Geisinger Clinic, Danville, PA 17822-2614. Tel.: 717-271-6682; Fax: 717-271-6701.

(^1)
The abbreviations used are: G proteins, a family of guanine nucleotide-binding proteins; G(s)alpha, the alpha subunit of G(s) involved in stimulation of adenylylcyclase; G(o)alpha, the alpha subunit of G(o); DTT, dithiothreitol; LPX, polyoxyethylene 10-lauryl ether; AMF, aluminum, magnesium, and fluoride.

(^2)
We have recently identified 3 new subunits, bringing the total number of known subunits to 10 (J. D. Robishaw, unpublished results).

(^3)
A paper reporting the specificity and functional consequences of prenylation of the (1) subunit expressed in the baculovirus system has been submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Jim Cali for construction of chimeras, Vivian Kalman and Marilyn Pray for baculovirus expression, Barbara Rabold for peptide synthesis, and Eric Balcueva for immuno-blotting. Also, we thank Dr. Carl Hansen and Dr. Howard Morgan for reading the manuscript and helpful suggestions.


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