- Email: [email protected]
[39] Preparation, characterization, and use of antibodies with specificity for G-protein y subunits
498
Gfly SUBUNITS
[39]
It is important not to expose the coverslips to UV light at this point. Rinse several times in sterile water and allow to dry. Sterilize under UV light for l0 min. The treated coverslips may be stored for 1 month at 4°. Warm to room temperature prior to plating cells. Acknowledgments We thank Dr. WilliamF. Simonds for providing unpublished information.This work supported by grants from the National Institutes of Health and Monsanto-Searle. N.G. is an Established Investigatorof the AmericanHeart Association.
[39] P r e p a r a t i o n , C h a r a c t e r i z a t i o n , a n d U s e o f A n t i b o d i e s w i t h S p e c i f i c i t y for G - P r o t e i n 7 S u b u n i t s
By JANET D. ROBISHAW and ERIC A. BALCUEVA Introduction The G proteins are composed of a, r , and 3' subunits, with each of these subunits having several different subtypes.~-3 Thus, the number of possible associations of these subunits to form functionally distinct G proteins is enormous. The production of subtype-specific antibodies is an important tool in determining the associations and functions of particular a, 8, and y subunits. The production of antibodies for the various a and fl subtypes of the G proteins was the focus of a chapter by Mumby and Gilman in a previous volume in this series. 4 Accordingly, in this chapter, we focus on the production of antipeptide antibodies for the different 3' subtypes and on special requirements for the use of these antibodies in detecting the various y subtypes. These antibodies have been shown to be specific and fairly versatile for use in detecting the various y subtypes by immunoblotting and immunostaining procedures.
I A. G. Gilman, Annu. Rev. Biochem. 56, 615 (1987). 2 M. I. Simon, M. P. Strathman, and N. Gautam, Science 252, 802 (1991). 3 j. j. Cali, E. A. Balcueva, I. Rybalkin, and J. D. Robishaw, J. Biol. Chem. 2,67, 24023 (1992). 4 S. M. Mumby and A. G. Gilman, this series, Vol. 195, p. 215.
METHODS IN ENZYMOLOGY, VOL. 237
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[39]
ANTIBODIES AGAINST G-PROTEIN 3/ SUBUNITS
499
Preparation of Antibodies
Selection o f Peptide Sequences When selecting a peptide that will produce an antibody of the desired specificity, the length and degree of conservation of the amino acid sequence are prime considerations. The minimum length of a peptide for antibody production is considered to be 10 amino acids. In this regard, a peptide based on a stretch of 8 amino acids that was conserved among the various 3/subunits failed to produce antibodies that recognized these proteins. Because conserved regions of the 7 subunits are limited to stretches of 8 amino acids or less, it has not been possible to produce antibodies that react indiscriminately with the majority of the 3' subunits, as has been possible for the a and/3 subunits of the G proteins. On the other hand, it has been possible to generate a series of antibodies that react specifically with each of the known 3' subunits by choosing peptide sequences from relatively nonconserved regions of these proteins (Fig. 1 and Table I). As hydrophilic amino acid residues are more likely to be exposed on the surface of proteins for recognition by the antipeptide antibodies, we have targeted regions containing an abundance of hydrophilic residues. In addition, we include regions containing proline residues, whenever possible, since they were found to be particularly useful in directing the specificity of the antibodies (e.g., B-17 and A-67 in Table I). When indicated, the peptides are synthesized with a cysteine residue at the amino terminus to facilitate conjugation to the carrier proteins (Table I). The peptides are synthesized using a solid-phase peptide synthesizer (Applied Biosystems, Foster City, CA; Model 430-1a), as described by the manufacturer. The purities of the peptides are assessed using C8 or C~8 reversed-phase high-performance liquid chromatography, and the sequences of the peptides are confirmed using amino acid analysis. Alternatively, when indicated, the peptides are synthesized on a heptalysine backbone, as described by Tam and co-workers5 (Table I).
Peptide Conjugation The peptides themselves are not particularly immunogenic, necessitating their conjugation to carrier proteins prior to injection. In general, peptides are conjugated to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO), as outlined by Green et al. 6 (Table I). For conjugation, 5 K. J. Chang, W. Pugh, S. G. Blanchard, J. McDermed, and J. P. Tam, Proc. Natl. Acad. Sci. U.S.A. 85, 4929 (1988). 6 N. H. Green, A. Alexander, S. Olson, T. Alexander, T. Shinnick, J. Sutcliff, and R. Lerner, Cell (Cambridge, Mass.) 28, 477 (1982).
500
Gfl7 SUBUNITS ............ MS
ATNN
I AQARK
[39] LVE
............. M S G S S S V A A M K K V V Q Q ~-M
I EAG
LRL
I E
EAGLN
K G E T P V N S T M S I G Q A R K M V E Q L K I E A S L C MA
~---M
Q LR
S NN
TA
S I A Q A R K
LVE
Q L KME
AN
I D
P V I N I E D L T E K D K L K M E V D Q L K K E V T L E
~-R
I K V S K A S S E L M S Y C E Q H A RN
D P L L V G V P A
~-R
V K V S Q A A A D L K Q F C L Q N A Q H D P L L T G V S S
~-R
I K V S K AAA
D L M T Y C D A H A C E D P L I T P V P T
~-R
I K V S K AAA
D L M A Y C E A H A K E D P L L T P V P A
~-R
M L V S K C C E E F R D Y V E E R S G E D P L V K G I P E
~7-S E N P F K D K K P - C I I L
7s-STNPrRPQXV-CSFL ~3-S E N P F R E K K F F C A I L ~2-S E N P F R E K K F F C A I L ~I-D K N P F K E L K G G C V I S
FIG. 1. Alignment of various y subunits. The protein sequences predicted from the cDNAs for YI, Y2, Y3, Ys, and 77 were aligned. Because the cDNA for Y4 has not been cloned, the protein sequence predicted from the polymerase chain reaction (PCR) fragment for Y4 has not been included. 2 The cDNAs for Y2 and 76 were cloned simultaneously in two different laboratories [N. Gautam, M. Baetscher, R. Aebersold, and M. I. Simon, Science 244, 971 (1989); J. D. Robishaw, V. K. Kalman, C. R. Moomaw, and C. A. Slaughter, J. Biol. Chem. 264, 15758 (1989)]. Since the sequences for Y2 and 76 were subsequently found to be identical, we have included only the sequence of Y2. Regions shown underlined correspond to peptides used for antibody production.
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Sigma) is used to link the peptides to KLH through cysteine residues. A detailed description of the conjugation procedure has been included previously in this series .4 By way of comparison, one of the peptides is also conjugated to tuberculin (purified protein derivative of tuberculin; Statens Seruminstitute, Copenhagen, Denmark), which has been reported to boost the antibody response to the peptide while giving rise to virtually no antibody response against itself. 7 In our hands, however, peptide conjugation to tuberculin 7 p. j. Lachman, L. Strangeways, A. Vyakarnam, and G. Evan, Ciba Found. Symp. 119, 25 (1986).
[39]
ANTIBODIES AGAINST G-PROTEIN ~/ SUBUNITS
501
TABLE I ANTIBODIES WITH SPECIFICITY FOR VARIOUS 'y SUBUNITS a
Antigen Designation
Yl 3'2
Y3 Y5 Y7
Region 37-49 2-14 2-14 35-46 35-49 48-61 48-62 48-61 2-17 50-63 46-59 46-60
Peptide
Antiserum Carrier
CEEFRDYVEERSG KLH CASNNTASIAQARK KLH ASNNTASIAQARK Heptalysine CDLMAYCEAHAK KLH DLMAYCEAHAKEDP Heptalysine CDPLLTPVPASENPF KLH DPLLTPVPASENPFR Heptalysine CDPLLTPVPASENPF Tuberculin CKGETPVNSTMSIGQAR KLH CTGVSSSTNPFRPQK KLH CDPLLVGVPASENPF KLH DPLLVGVPASENPFK Heptalysine
Reactivity 3 of 3 1 of 4 0 of 2 2 of 4 1 of 2 4 of 4 2 of 2 1 of 2 1 of 2 2 of 2 2 of 2 1 of 2
Specificity Code Yl Y2
A-4 A-75
Y2 Y2 3'2 ~ Y3 Y2 ~ Y3 Y2 ~ Y3 Y3 Y5 Y7 Y7
A-25 B-60 B-17 B-63 B-23 B-53 D-9 A-67 B-65
a The region numbers indicate the location of the peptide sequences in the various y subunits. The reactivity refers to the number of rabbits that reacted with the protein of interest by immunoblotting out of the total number of rabbits that were immunized with the peptide. The code refers to the numbers assigned to the rabbits by the investigators (J.D.R. and E.A.B.).
results in a lower proportion o f rabbits that produce antibodies to the protein o f interest and no i m p r o v e m e n t in the titer of the antibodies produced (Table I). Also, by w a y of comparison, three of the peptides are attached to heptalysine b a c k b o n e s , which has b e e n reported to enhance the antibody response by presenting multiple copies of the peptide on each heptalysine b a c k b o n e : Again, however, this p r o c e d u r e results in no i m p r o v e m e n t in the proportion of rabbits that produce antibodies to the proteins and no i m p r o v e m e n t in the titer of antibodies p r o d u c e d (Table I). G i v e n the greater degree of difficulty in synthesizing peptides on the heptalysine b a c k b o n e and the lack of i m p r o v e m e n t in the titers o f the antibodies produced, we do not generally r e c o m m e n d the use of the latter procedure.
Immunization and Bleeding Schedule E a c h o f the conjugated or heptalysine peptides (equivalent to 200/.~g o f peptide) is solubilized with 500/zl of phosphate-buffered saline (PBS), emulsified with 500/xl o f F r e u n d ' s complete adjuvant ( G I B C O L a b o r a tories, G r a n d Island, NY), and administered to N e w Zealand White rabbits b y subcutaneous injection at four sites on the back. T w o weeks after the first injection, the same amount of conjugated peptide is emulsified with F r e u n d ' s incomplete adjuvant and administered to rabbits by subcutane-
502
G/3y soauNn-s
[39]
ous injection. One week after the second injection, the same amount of conjugated peptide is adsorbed to alum, a as described in detail elsewhere, 4,8 and administered to rabbits by intraperitoneal injection at two sites in the abdomen. In general, this immunization protocol results in the production of antibodies that recognize the peptides and, in most cases, the proteins of interest. However, the titer and specificity of the antibodies produced are found to vary widely between rabbits. Thus, whenever possible, we recommend that each of the conjugated peptides be injected into two or more rabbits. Two conjugated peptides are emulsified with RIBI adjuvant in place of Freund's complete and incomplete adjuvants. The RIBI adjuvant (RIBI Immunochem Research, Inc., Hamilton, MT) has been reported to induce a strong antibody response, without causing the granuloma formation that is a frequent side effect of the Freund's adjuvant. The rabbits receiving the conjugated peptides with the Freund's adjuvant produce antibodies that react specifically with either the/31 or the/32 subunit (three out of four rabbits in each case). In contrast, the rabbits receiving the conjugated peptides with the RIBI adjuvant produce antibodies of similar titers that react indiscriminately with both the/31 and/3z subunits. Thus, the use of RIBI adjuvant adversely affects the selectivity of the antibody response to peptides based on the sequences of two closely related proteins. Although the applicability of this finding to other closely related proteins, such as the 3, subunits, is not known, the use of RIBI adjuvant is not generally recommended for producing antibodies to closely related proteins in a limited number of rabbits. Beginning 1 week after the final injection, rabbits are bled every 2 weeks to monitor antibody production. The blood ( - 2 0 ml) is allowed to clot for 1 hr at 37°, and the clot is allowed to retract for several hours at 4°. Following removal from the clot, the serum is centrifuged at 10,000 g for 10 min at 4° to remove residual blood cells, then divided into aliquots for storage at - 80°. To monitor the presence of antibodies, the serum is tested for reactivity to the peptide by an enzyme-linked immunosorbent assay (ELISA) procedure. 9 If desired, antibodies to the carrier protein are effectively removed by passage through a peptide affinity column. 4 Immune Response as Monitored by Immunoblotting Because discrepancies between antibody titers for the peptide versus the protein of interest have been observed, the serum is routinely tested a C. A. Williams and M. W. Chase, Methods lmmunol. Immunochem. 1, 201 (1967). 9 E. Engvali, this series, Vol. 70, p. 419.
[39]
ANTIBODIES AGAINST G-PROTEIN 'y SUBUNITS
503
for reactivity to the purified protein by an immunoblotting procedure. 1° The transfer and immunoblotting procedures are presented in some detail, since both of these procedures have been modified to optimize the detection of the y subunits. 11 Purification of a mixture of the fly subunits is carded out from bovine brain, as described previously. 12 The different y subunits are resolved on a 15% polyacrylamide separating gel (acrylamide to bisacrylamide ratio of 29 : 1) by sodium dodecyl suifate-polyacrylamide gel electrophoresis (SDS-PAGE). In general, 2 /~g of purified fly subunits (equivalent to -300 ng of purified y subunit) is loaded on each lane of the gel. After electrophoresis, the proteins are transferred to Nitroplus nitrocellulose (0.45/.~m pore size; Micron Separations, Inc., Westboro, MA), using the Hoeffer Transphor system (TE series, Hoefer Scientific Instruments, San Francisco, CA). Transfer is carried out at 30 V overnight in transfer buffer [25 mM Tris, 190 mM glycine, and 20% (v/v) methanol, pH 8.2] that had been heated to 70 °, using a circulating water bath (Forma Scientific, Model 2006). As shown in Fig. 2, heating the transfer buffer results in a greater than 20-fold increase in the sensitivity of detection of the y subunits by immunoblotting, it presumably by maintaining the proteins in a denatured state during transfer and/or binding to the nitrocellulose. Denaturation of the y subunits would be expected to have a beneficial effect, since antipeptide antibodies generally recognize the epitopes more effectively in denatured proteins than in native proteins. In contrast, inclusion of the protein denaturant SDS in the transfer buffer or heating the transferred proteins on the nitrocellulose by baking or autoclaving are not nearly so effective in enhancing the sensitivity of detection of the y subunits by immunoblotting (Fig. 2). Another factor found to enhance the sensitivity of detection of the y subunits by immunoblotting is the type of paper used for the transfer. Of the various types of paper tried, the Nitroplus nitrocellulose and the diazotized papers (aminobenzoyloxymethyl cellulose and aminophenylthioether cellulose; Schleicher and Schuell, Inc., Keene, NH) have been found to exhibit a 5-fold higher binding capacity than nitrocellulose (Schleicher and Schuell) and Immobilon (Millipore, Inc., Bedford, MA). Although Nitroplus nitrocellulose and diazotized papers are found to have similar binding capacities, the use of the Nitroplus nitrocellulose is favored by the comparatively lower cost and longer shelf life of this paper. 10 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 11 j. D. Robishaw and E. A. Balcueva, Anal. Biochem. 208, 283 (1993). 12 p. C. Sternweis and J. D. Robishaw, J. Biol. Chem. 259, 13806 (1984).
G/3y SUBUNITS
504
[39]
B Transfer at 70°C
A Transfer at 25°C
DDD
- - 14.3 k D a - --6.5
kDa - -
"O
"0
•= D
~
0
m
m
,<
O
II]
<
FIG. 2. Effect of high-temperature transfer to greatly enhance detection of G-protein y subunits with antipeptide antibodies. Solubilized bovine brain membrane proteins (100/zg brain) and partially purified bovine brain G protein standard (10 t~g Std) were resolved on a 15% polyacrylamide gel. After transfer of the resolved proteins to nitrocellulose, the blots were air-dried, baked in v a c u o at 80°, or autoclaved at 120°. The nitrocellulose pieces were incubated with anti-y7 antibody (A-67), as the primary antibody, and t25I-labeled goat antirabbit antibody, as the secondary antibody. To obtain autoradiographic exposures, the nitrocellulose pieces were exposed to film for 18 hr. Numbers between (A) and (B) indicate apparent molecular masses, based on the mobilities of aprotinin and lysozyme standards. (Reproduced by permission from Ref. 11.)
To determine the efficiency of transfer, the Nitroplus nitrocellulose blots are stained with Ponceau S or amido black (Sigma) prior to immunological processing. For processing, the nitrocellulose blots are incubated for 1 hr in high-detergent buffer A [50 mM Tris (pH 8), 2 mM CaCI2, 80 mM NaC1, 5% nonfat dry milk, 2% Nonidet P-40 (NP-40) and 0.2% SDS] with the appropriate antipeptide antibodies? After three 5-rain washes in high-detergent buffer A the nitrocellulose blots are incubated for 1 hr in high-detergent buffer A with ~25I-labeled goat anti-rabbit F(ab')2 fragment [1 x 106 disintegrations/min (dpm)/ml; New England Nuclear, Boston, MA]. After three 5-rain washes in high-detergent buffer A followed by three 5-min washes in detergent-free buffer A, the nitrocellulose blots are air-dried at room temperature. The incubation and wash times are minimized to prevent loss of the low molecular weight y subunits during immunological processing. To obtain autoradiographic images, the nitrocellulose blots are exposed with an intensifying screen to Kodak (Rochester, NY) XAR-5 film overnight. If desired, the intensities of the immunodetectable bands are quantitated by scanning the nitrocellulose blots with the AMBIS Radioanalytic Imaging System (AMBIS Inc., San Diego, CA).
[39]
ANTIBODIES AGAINST G-PROTEIN 'y SUBUNITS
505
anti -'~ anti "'~z anti "'Y3 l
!
anti "'~s anti "'Y7 FIG. 3. Specificity of antibodies for recombinant ~/subunits expressed in Sf9 cells. Cholate extracts of particulate fractions from Sf9 cells infected with recombinant viruses encoding the indicated 7 subunits or the wild-type virus (100 /~g) were subjected to SDS-PAGE followed by immunoblot analysis with the various antibodies. Antibodies used for blotting were the anti-y1 antibody A-4, the anti-y2 antibody A-75, the anti-% antibody B-53, the anti315antibody D-9, and the anti-~/7 antibody A-67.
Characterization of Antibody Specificity
The specificity of the antipeptide antibodies for the various 7 subunits is best demonstrated by immunoblotting proteins of the appropriate sizes in cholate extracts from Sf9 cells (Spodopterafrugiperda ovary) infected with recombinant baculoviruses encoding the various 7 subunits)'~3 As shown in Fig. 3, the cholate-soluble membrane extracts from Sf9 cells infected with recombinant baculoviruses encoding the 71,72, 73,75, and 77 subunits were resolved on a 15% polyacrylamide-SDS gel, transferred to nitrocellulose, and then immunoblotted. As expected, each of the antibodies generated against a peptide based on sequence unique to the 71, 72, 73, 75, and 77 subunits recognized a protein of the appropriate size (5-7 kDa range) only in the cholate-soluble membrane extract from cells infected with the virus for the corresponding 7 subunit. The specificity of each of these antibodies was further demonstrated by the failure of each of these antibodies to detect similarly sized proteins in extracts from cells infected with viruses for the noncorresponding 7 subunits, or with the wild-type virus. 13j. D. Robishaw, V. K. Kalman, and K. L. Proulx, Biochem. J. 286, 672 (1992).
506
G/3y SUBUNITS
[39]
Usefulness of Antibodies
With the production of antibodies to the different y subtypes, it is now possible to determine the distribution of the y subtypes within various tissues, to examine the localization of the y subtypes within particular cell types, and to follow the synthesis, posttranslational processing, and assembly of particular a,/3, and y subunits of the G proteins. This information is ultimately needed to determine the role of specific combinations of a/3y subunits of the G proteins in a large number of different receptormediated signaling pathways. Tissue Distribution and Localization o f y Subunits The identification of the 3' subunits in a tissue or cell type is greatly facilitated by the use of subtype-specific antibodies) Such studies have lagged behind those with the a and/3 subunits owing to the inability of antibodies against the y subunits to detect these proteins in whole cell or membrane extracts by immunoblotting. As discussed above, by incorporating a high-temperature transfer step to allow the antipeptide antibodies to gain access to the epitopes, it has become possible for the first time to utilize these antibodies to determine the tissue distribution of the y subunits. The preparation and solubilization of membranes from several bovine tissues with 0.9% cholate have been described previously) In contrast to the/3 subtypes, the various y subtypes showed a more selective pattern of expression. Thus, the Yz and Y3 subunits were preferentially expressed in brain, whereas the Y5 and "Y7subunits were widely expressed in brain, heart, kidney, spleen, liver, and lung (Fig. 4). The Yl subunit was exclusively expressed in the retina (data not shown). This pattern of distribution for the 3' subunits is reminiscent of that for the o~ subunits, that is, if not found largely in the brain or retina, the a subunits were found in a variety of tissues. Ascertainment of the tissue distribution of specific subunits will be of importance in determining which of the large number of possible a/3y combinatorial associations actually occur in a physiological setting. The subtype-specific antibodies have also been successfully used for the cellular localization of the y subunits in monkey retina, 14 which contains readily distinguishable cell types with highly specialized functions. The/31 and Yl subunits were found to be localized in the rod outer segments (ROS), whereas the/33 and "Y2subunits were found to be localized in the cone outer segments. It is likely that the localization of particular a as 14 y . W. Peng, J. D. Robishaw, M. A. Levine, and K. W. Yau, Proc. Natl. Acad. Sci. U.S.A. 89, 10882 (1992).
[39]
ANTIBODIES
AGAINST
G-PROTEIN
'y S U B U N I T S
507
"o t__ "10
~
.t,-.
m
tl~
Idea
6.5~
%
6.5 m
3's
e.5
3'a
6"5m
'~'2 1
2
3
4
5
6
7
FIG. 4. Tissue distribution of y subunits. Cholate extracts of particulate fraction~ from the indicated tissues (100/.tg) were subjected to SDS-PAGE and immunoblot analysis, as described in the text. The standard shown in lane l represents 4/~g of purified bovine brain G proteins. The 6.5-kDa molecular mass marker was aprotinin. Exposure times were 2 and 4 days for the Y2and "/3 blots, respectively, and 18 hr for the Y5and ')17blots.
well as fly subunits within different cell types in the retina reflects the association of these subunits into different G proteins, with the phototransduction properties characteristic of the rod and cone outer segments. A similar a p p r o a c h to colocalize a , / 3 , and Y subunits in specific cell types in other tissues m a y provide a useful w a y of defining the n u m b e r o f possible a[3y associations that can o c c u r in a physiological setting. Processing o f y Subunits
The subtype-specific antibodies have also b e e n used to examine the posttranslational processing of the y subunits, which first involves the a t t a c h m e n t of a prenyl group to a cysteine residue near the carboxyl terminus of these proteins. 15,16 Insect (Sf9) cells expressing the Y2 and 3'3 subunits were fractionated into cytosolic and particulate fractions. Following extraction with 0.9% cholate, the particulate fractions were then frac15w. A. Maltese and J. D. Robishaw, J. Biol. Chem. 265, 18071 (1990). 16S. M. Mumby, P. J. Casey, A. G. Gilman, S. Gutowski, and P. C. Sternweis, Proc. Natl. Acad. Sci. U.S.A. 87, 5873 (1990).
508
Gfl~/ SUBUNITS
[39]
tionated into cholate-soluble and cholate-insoluble particulate fractions. As shown in Fig. 5, the nonprenylated forms of the 3,2 and 3,3 subunits, which migrated m o r e quickly on the gel, 13 were expressed largely in the cytosolic and insoluble particulate fractions, respectively (compare u p p e r and lower portions of Fig. 5). Although the reason for the difference in subcellular localization is not known, the localization of the 3'3 subunit in the insoluble particulate fraction m a y reflect an increased tendency of the 3'3 subunit to aggregate on overexpression. Conversely, the prenylated forms of the 3"2 and 3'3 subunits, which migrated m o r e slowly, 13 were e x p r e s s e d exclusively in the soluble and insoluble particulate fractions. The origin of a third f o r m of the 3'3 subunit in the insoluble particulate fraction has not b e e n determined, but it appears to represent an aggregated f o r m of the 3"3 subunit. Nevertheless, the localization of the prenylated
3'=-Infected
3'=,8=-Infected kDa
:Da
%-Infected
%~6~-Infected
%~=-Infected kDa
:Da
FIG. 5. Expression and subcellular distribution of various combinations of the ill, t2, ~t2, and )t3 subunits of the G proteins in Sf9 cells. At 72 hr after infection with various combinations of viruses encoding the ill, BE, 3t2, and 73 subunits, cells were fractionated to yield the cytosolic (C), cholate-soluble particulate (SP), and cholate-insoluble particulate
(IP) fractions. The same percentage 05%) of each fraction was resolved by SDS-PAGE on a 15% polyacrylamide gel, then transferred to nitrocellulose blots. The nitrocellulose blots were incubated with either the 72 antibody B-17 (top) or the 73 antibody B-53 (bottom), using ~2S-labeledgoat anti-rabbit secondary antibody for detection. The blots were exposed to film for 2 days with an intensifying screen.
[40]
ISOPRENYLATION
OF
G3,
AND G-PROTEIN
EFFECTORS
509
forms of the 3'2 and 3'3 subunits in the soluble particulate fraction is consistent with the role of prenylation in promoting membrane interaction. 17,18 Interestingly, the proportion of the prenylated forms of the 3'2 and 3'3 subunits was significantly increased by coexpression of the fll or/32 subunits in insect cells, 13 as demonstrated by an increase in the amount of the slower migrating forms in the soluble particulate fractions from/33'infected cells compared to 3,-infected cells (Fig. 5). Although the mechanism of this effect is not known, association of the/3 and 3' subunits into a functional heterodimer does not appear to be required for two reasons. First, the 3, subunit is able to undergo prenylation when expressed alone. Second, both the/31 and/32 subunits are able to enhance prenylation of the 3'3 subunit even though a number of investigators have shown that the /32 and 3"3 subunits do not associate to form a functional dimer. 19-2~ Acknowledgments This work was supported by National Institutes of Health Grant GM 39867 and an American Heart Association Established Investigatorship to J.D.R.
17 W. F. Simonds, J. E. Butrynski, N. Gautam, C. G. Unson, and A. M. Spiegel, J. Biol. Chem. 266, 5363 (1991). ta K. H. Muntz, P. C. Sternweis, A. G. Gilman, and S. M. Mumby, Mol. Biol. Cell3, 49 (1992). 19j. Iniguez-Lluhi, M. Simon, J. D. Robishaw, and A. G. Gilman, J. Biol. Chem. 7,67, 23409 (1992). 20 C. J. Schmidt, T. C. Thomas, M. A. Levine, and E. J. Neer, J. Biol. Chem. 1,67, 13807 (1992). 21 A. N. Pronin and N. Gautam, Proc. Natl. Acad. Sci. U.S.A. 89, 6220 (1992).
[40] I s o p r e n y l a t i o n o f y S u b u n i t s a n d G - P r o t e i n E f f e c t o r s
By
BERNARD K . - K . F U N G , JANMEET S. A N A N T , W U N - C H E N L I N , OLIVIA C. O N G , a n d HARVEY K . YAMANE
Introduction
Protein isoprenylation is a recently discovered posttranslational modification.1 The covalent addition of a isoprenyl moiety to a polypeptide was first described in the yeast mating factors. 2 These short polypeptides, I S. Clarke, Annu. Rev. Biochem. 61, 355 (1992). 2 y. Kamiya, A. Sakurai, S. Tamura, and N. Takahashi, Biochem. Biophys. Res. Commun. 83, 1077 (1973).
METHODS IN ENZYMOLOGY, VOL. 237
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
Gfly SUBUNITS
[39]
It is important not to expose the coverslips to UV light at this point. Rinse several times in sterile water and allow to dry. Sterilize under UV light for l0 min. The treated coverslips may be stored for 1 month at 4°. Warm to room temperature prior to plating cells. Acknowledgments We thank Dr. WilliamF. Simonds for providing unpublished information.This work supported by grants from the National Institutes of Health and Monsanto-Searle. N.G. is an Established Investigatorof the AmericanHeart Association.
[39] P r e p a r a t i o n , C h a r a c t e r i z a t i o n , a n d U s e o f A n t i b o d i e s w i t h S p e c i f i c i t y for G - P r o t e i n 7 S u b u n i t s
By JANET D. ROBISHAW and ERIC A. BALCUEVA Introduction The G proteins are composed of a, r , and 3' subunits, with each of these subunits having several different subtypes.~-3 Thus, the number of possible associations of these subunits to form functionally distinct G proteins is enormous. The production of subtype-specific antibodies is an important tool in determining the associations and functions of particular a, 8, and y subunits. The production of antibodies for the various a and fl subtypes of the G proteins was the focus of a chapter by Mumby and Gilman in a previous volume in this series. 4 Accordingly, in this chapter, we focus on the production of antipeptide antibodies for the different 3' subtypes and on special requirements for the use of these antibodies in detecting the various y subtypes. These antibodies have been shown to be specific and fairly versatile for use in detecting the various y subtypes by immunoblotting and immunostaining procedures.
I A. G. Gilman, Annu. Rev. Biochem. 56, 615 (1987). 2 M. I. Simon, M. P. Strathman, and N. Gautam, Science 252, 802 (1991). 3 j. j. Cali, E. A. Balcueva, I. Rybalkin, and J. D. Robishaw, J. Biol. Chem. 2,67, 24023 (1992). 4 S. M. Mumby and A. G. Gilman, this series, Vol. 195, p. 215.
METHODS IN ENZYMOLOGY, VOL. 237
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[39]
ANTIBODIES AGAINST G-PROTEIN 3/ SUBUNITS
499
Preparation of Antibodies
Selection o f Peptide Sequences When selecting a peptide that will produce an antibody of the desired specificity, the length and degree of conservation of the amino acid sequence are prime considerations. The minimum length of a peptide for antibody production is considered to be 10 amino acids. In this regard, a peptide based on a stretch of 8 amino acids that was conserved among the various 3/subunits failed to produce antibodies that recognized these proteins. Because conserved regions of the 7 subunits are limited to stretches of 8 amino acids or less, it has not been possible to produce antibodies that react indiscriminately with the majority of the 3' subunits, as has been possible for the a and/3 subunits of the G proteins. On the other hand, it has been possible to generate a series of antibodies that react specifically with each of the known 3' subunits by choosing peptide sequences from relatively nonconserved regions of these proteins (Fig. 1 and Table I). As hydrophilic amino acid residues are more likely to be exposed on the surface of proteins for recognition by the antipeptide antibodies, we have targeted regions containing an abundance of hydrophilic residues. In addition, we include regions containing proline residues, whenever possible, since they were found to be particularly useful in directing the specificity of the antibodies (e.g., B-17 and A-67 in Table I). When indicated, the peptides are synthesized with a cysteine residue at the amino terminus to facilitate conjugation to the carrier proteins (Table I). The peptides are synthesized using a solid-phase peptide synthesizer (Applied Biosystems, Foster City, CA; Model 430-1a), as described by the manufacturer. The purities of the peptides are assessed using C8 or C~8 reversed-phase high-performance liquid chromatography, and the sequences of the peptides are confirmed using amino acid analysis. Alternatively, when indicated, the peptides are synthesized on a heptalysine backbone, as described by Tam and co-workers5 (Table I).
Peptide Conjugation The peptides themselves are not particularly immunogenic, necessitating their conjugation to carrier proteins prior to injection. In general, peptides are conjugated to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO), as outlined by Green et al. 6 (Table I). For conjugation, 5 K. J. Chang, W. Pugh, S. G. Blanchard, J. McDermed, and J. P. Tam, Proc. Natl. Acad. Sci. U.S.A. 85, 4929 (1988). 6 N. H. Green, A. Alexander, S. Olson, T. Alexander, T. Shinnick, J. Sutcliff, and R. Lerner, Cell (Cambridge, Mass.) 28, 477 (1982).
500
Gfl7 SUBUNITS ............ MS
ATNN
I AQARK
[39] LVE
............. M S G S S S V A A M K K V V Q Q ~-M
I EAG
LRL
I E
EAGLN
K G E T P V N S T M S I G Q A R K M V E Q L K I E A S L C MA
~---M
Q LR
S NN
TA
S I A Q A R K
LVE
Q L KME
AN
I D
P V I N I E D L T E K D K L K M E V D Q L K K E V T L E
~-R
I K V S K A S S E L M S Y C E Q H A RN
D P L L V G V P A
~-R
V K V S Q A A A D L K Q F C L Q N A Q H D P L L T G V S S
~-R
I K V S K AAA
D L M T Y C D A H A C E D P L I T P V P T
~-R
I K V S K AAA
D L M A Y C E A H A K E D P L L T P V P A
~-R
M L V S K C C E E F R D Y V E E R S G E D P L V K G I P E
~7-S E N P F K D K K P - C I I L
7s-STNPrRPQXV-CSFL ~3-S E N P F R E K K F F C A I L ~2-S E N P F R E K K F F C A I L ~I-D K N P F K E L K G G C V I S
FIG. 1. Alignment of various y subunits. The protein sequences predicted from the cDNAs for YI, Y2, Y3, Ys, and 77 were aligned. Because the cDNA for Y4 has not been cloned, the protein sequence predicted from the polymerase chain reaction (PCR) fragment for Y4 has not been included. 2 The cDNAs for Y2 and 76 were cloned simultaneously in two different laboratories [N. Gautam, M. Baetscher, R. Aebersold, and M. I. Simon, Science 244, 971 (1989); J. D. Robishaw, V. K. Kalman, C. R. Moomaw, and C. A. Slaughter, J. Biol. Chem. 264, 15758 (1989)]. Since the sequences for Y2 and 76 were subsequently found to be identical, we have included only the sequence of Y2. Regions shown underlined correspond to peptides used for antibody production.
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Sigma) is used to link the peptides to KLH through cysteine residues. A detailed description of the conjugation procedure has been included previously in this series .4 By way of comparison, one of the peptides is also conjugated to tuberculin (purified protein derivative of tuberculin; Statens Seruminstitute, Copenhagen, Denmark), which has been reported to boost the antibody response to the peptide while giving rise to virtually no antibody response against itself. 7 In our hands, however, peptide conjugation to tuberculin 7 p. j. Lachman, L. Strangeways, A. Vyakarnam, and G. Evan, Ciba Found. Symp. 119, 25 (1986).
[39]
ANTIBODIES AGAINST G-PROTEIN ~/ SUBUNITS
501
TABLE I ANTIBODIES WITH SPECIFICITY FOR VARIOUS 'y SUBUNITS a
Antigen Designation
Yl 3'2
Y3 Y5 Y7
Region 37-49 2-14 2-14 35-46 35-49 48-61 48-62 48-61 2-17 50-63 46-59 46-60
Peptide
Antiserum Carrier
CEEFRDYVEERSG KLH CASNNTASIAQARK KLH ASNNTASIAQARK Heptalysine CDLMAYCEAHAK KLH DLMAYCEAHAKEDP Heptalysine CDPLLTPVPASENPF KLH DPLLTPVPASENPFR Heptalysine CDPLLTPVPASENPF Tuberculin CKGETPVNSTMSIGQAR KLH CTGVSSSTNPFRPQK KLH CDPLLVGVPASENPF KLH DPLLVGVPASENPFK Heptalysine
Reactivity 3 of 3 1 of 4 0 of 2 2 of 4 1 of 2 4 of 4 2 of 2 1 of 2 1 of 2 2 of 2 2 of 2 1 of 2
Specificity Code Yl Y2
A-4 A-75
Y2 Y2 3'2 ~ Y3 Y2 ~ Y3 Y2 ~ Y3 Y3 Y5 Y7 Y7
A-25 B-60 B-17 B-63 B-23 B-53 D-9 A-67 B-65
a The region numbers indicate the location of the peptide sequences in the various y subunits. The reactivity refers to the number of rabbits that reacted with the protein of interest by immunoblotting out of the total number of rabbits that were immunized with the peptide. The code refers to the numbers assigned to the rabbits by the investigators (J.D.R. and E.A.B.).
results in a lower proportion o f rabbits that produce antibodies to the protein o f interest and no i m p r o v e m e n t in the titer of the antibodies produced (Table I). Also, by w a y of comparison, three of the peptides are attached to heptalysine b a c k b o n e s , which has b e e n reported to enhance the antibody response by presenting multiple copies of the peptide on each heptalysine b a c k b o n e : Again, however, this p r o c e d u r e results in no i m p r o v e m e n t in the proportion of rabbits that produce antibodies to the proteins and no i m p r o v e m e n t in the titer of antibodies p r o d u c e d (Table I). G i v e n the greater degree of difficulty in synthesizing peptides on the heptalysine b a c k b o n e and the lack of i m p r o v e m e n t in the titers o f the antibodies produced, we do not generally r e c o m m e n d the use of the latter procedure.
Immunization and Bleeding Schedule E a c h o f the conjugated or heptalysine peptides (equivalent to 200/.~g o f peptide) is solubilized with 500/zl of phosphate-buffered saline (PBS), emulsified with 500/xl o f F r e u n d ' s complete adjuvant ( G I B C O L a b o r a tories, G r a n d Island, NY), and administered to N e w Zealand White rabbits b y subcutaneous injection at four sites on the back. T w o weeks after the first injection, the same amount of conjugated peptide is emulsified with F r e u n d ' s incomplete adjuvant and administered to rabbits by subcutane-
502
G/3y soauNn-s
[39]
ous injection. One week after the second injection, the same amount of conjugated peptide is adsorbed to alum, a as described in detail elsewhere, 4,8 and administered to rabbits by intraperitoneal injection at two sites in the abdomen. In general, this immunization protocol results in the production of antibodies that recognize the peptides and, in most cases, the proteins of interest. However, the titer and specificity of the antibodies produced are found to vary widely between rabbits. Thus, whenever possible, we recommend that each of the conjugated peptides be injected into two or more rabbits. Two conjugated peptides are emulsified with RIBI adjuvant in place of Freund's complete and incomplete adjuvants. The RIBI adjuvant (RIBI Immunochem Research, Inc., Hamilton, MT) has been reported to induce a strong antibody response, without causing the granuloma formation that is a frequent side effect of the Freund's adjuvant. The rabbits receiving the conjugated peptides with the Freund's adjuvant produce antibodies that react specifically with either the/31 or the/32 subunit (three out of four rabbits in each case). In contrast, the rabbits receiving the conjugated peptides with the RIBI adjuvant produce antibodies of similar titers that react indiscriminately with both the/31 and/3z subunits. Thus, the use of RIBI adjuvant adversely affects the selectivity of the antibody response to peptides based on the sequences of two closely related proteins. Although the applicability of this finding to other closely related proteins, such as the 3, subunits, is not known, the use of RIBI adjuvant is not generally recommended for producing antibodies to closely related proteins in a limited number of rabbits. Beginning 1 week after the final injection, rabbits are bled every 2 weeks to monitor antibody production. The blood ( - 2 0 ml) is allowed to clot for 1 hr at 37°, and the clot is allowed to retract for several hours at 4°. Following removal from the clot, the serum is centrifuged at 10,000 g for 10 min at 4° to remove residual blood cells, then divided into aliquots for storage at - 80°. To monitor the presence of antibodies, the serum is tested for reactivity to the peptide by an enzyme-linked immunosorbent assay (ELISA) procedure. 9 If desired, antibodies to the carrier protein are effectively removed by passage through a peptide affinity column. 4 Immune Response as Monitored by Immunoblotting Because discrepancies between antibody titers for the peptide versus the protein of interest have been observed, the serum is routinely tested a C. A. Williams and M. W. Chase, Methods lmmunol. Immunochem. 1, 201 (1967). 9 E. Engvali, this series, Vol. 70, p. 419.
[39]
ANTIBODIES AGAINST G-PROTEIN 'y SUBUNITS
503
for reactivity to the purified protein by an immunoblotting procedure. 1° The transfer and immunoblotting procedures are presented in some detail, since both of these procedures have been modified to optimize the detection of the y subunits. 11 Purification of a mixture of the fly subunits is carded out from bovine brain, as described previously. 12 The different y subunits are resolved on a 15% polyacrylamide separating gel (acrylamide to bisacrylamide ratio of 29 : 1) by sodium dodecyl suifate-polyacrylamide gel electrophoresis (SDS-PAGE). In general, 2 /~g of purified fly subunits (equivalent to -300 ng of purified y subunit) is loaded on each lane of the gel. After electrophoresis, the proteins are transferred to Nitroplus nitrocellulose (0.45/.~m pore size; Micron Separations, Inc., Westboro, MA), using the Hoeffer Transphor system (TE series, Hoefer Scientific Instruments, San Francisco, CA). Transfer is carried out at 30 V overnight in transfer buffer [25 mM Tris, 190 mM glycine, and 20% (v/v) methanol, pH 8.2] that had been heated to 70 °, using a circulating water bath (Forma Scientific, Model 2006). As shown in Fig. 2, heating the transfer buffer results in a greater than 20-fold increase in the sensitivity of detection of the y subunits by immunoblotting, it presumably by maintaining the proteins in a denatured state during transfer and/or binding to the nitrocellulose. Denaturation of the y subunits would be expected to have a beneficial effect, since antipeptide antibodies generally recognize the epitopes more effectively in denatured proteins than in native proteins. In contrast, inclusion of the protein denaturant SDS in the transfer buffer or heating the transferred proteins on the nitrocellulose by baking or autoclaving are not nearly so effective in enhancing the sensitivity of detection of the y subunits by immunoblotting (Fig. 2). Another factor found to enhance the sensitivity of detection of the y subunits by immunoblotting is the type of paper used for the transfer. Of the various types of paper tried, the Nitroplus nitrocellulose and the diazotized papers (aminobenzoyloxymethyl cellulose and aminophenylthioether cellulose; Schleicher and Schuell, Inc., Keene, NH) have been found to exhibit a 5-fold higher binding capacity than nitrocellulose (Schleicher and Schuell) and Immobilon (Millipore, Inc., Bedford, MA). Although Nitroplus nitrocellulose and diazotized papers are found to have similar binding capacities, the use of the Nitroplus nitrocellulose is favored by the comparatively lower cost and longer shelf life of this paper. 10 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 11 j. D. Robishaw and E. A. Balcueva, Anal. Biochem. 208, 283 (1993). 12 p. C. Sternweis and J. D. Robishaw, J. Biol. Chem. 259, 13806 (1984).
G/3y SUBUNITS
504
[39]
B Transfer at 70°C
A Transfer at 25°C
DDD
- - 14.3 k D a - --6.5
kDa - -
"O
"0
•= D
~
0
m
m
,<
O
II]
<
FIG. 2. Effect of high-temperature transfer to greatly enhance detection of G-protein y subunits with antipeptide antibodies. Solubilized bovine brain membrane proteins (100/zg brain) and partially purified bovine brain G protein standard (10 t~g Std) were resolved on a 15% polyacrylamide gel. After transfer of the resolved proteins to nitrocellulose, the blots were air-dried, baked in v a c u o at 80°, or autoclaved at 120°. The nitrocellulose pieces were incubated with anti-y7 antibody (A-67), as the primary antibody, and t25I-labeled goat antirabbit antibody, as the secondary antibody. To obtain autoradiographic exposures, the nitrocellulose pieces were exposed to film for 18 hr. Numbers between (A) and (B) indicate apparent molecular masses, based on the mobilities of aprotinin and lysozyme standards. (Reproduced by permission from Ref. 11.)
To determine the efficiency of transfer, the Nitroplus nitrocellulose blots are stained with Ponceau S or amido black (Sigma) prior to immunological processing. For processing, the nitrocellulose blots are incubated for 1 hr in high-detergent buffer A [50 mM Tris (pH 8), 2 mM CaCI2, 80 mM NaC1, 5% nonfat dry milk, 2% Nonidet P-40 (NP-40) and 0.2% SDS] with the appropriate antipeptide antibodies? After three 5-rain washes in high-detergent buffer A the nitrocellulose blots are incubated for 1 hr in high-detergent buffer A with ~25I-labeled goat anti-rabbit F(ab')2 fragment [1 x 106 disintegrations/min (dpm)/ml; New England Nuclear, Boston, MA]. After three 5-rain washes in high-detergent buffer A followed by three 5-min washes in detergent-free buffer A, the nitrocellulose blots are air-dried at room temperature. The incubation and wash times are minimized to prevent loss of the low molecular weight y subunits during immunological processing. To obtain autoradiographic images, the nitrocellulose blots are exposed with an intensifying screen to Kodak (Rochester, NY) XAR-5 film overnight. If desired, the intensities of the immunodetectable bands are quantitated by scanning the nitrocellulose blots with the AMBIS Radioanalytic Imaging System (AMBIS Inc., San Diego, CA).
[39]
ANTIBODIES AGAINST G-PROTEIN 'y SUBUNITS
505
anti -'~ anti "'~z anti "'Y3 l
!
anti "'~s anti "'Y7 FIG. 3. Specificity of antibodies for recombinant ~/subunits expressed in Sf9 cells. Cholate extracts of particulate fractions from Sf9 cells infected with recombinant viruses encoding the indicated 7 subunits or the wild-type virus (100 /~g) were subjected to SDS-PAGE followed by immunoblot analysis with the various antibodies. Antibodies used for blotting were the anti-y1 antibody A-4, the anti-y2 antibody A-75, the anti-% antibody B-53, the anti315antibody D-9, and the anti-~/7 antibody A-67.
Characterization of Antibody Specificity
The specificity of the antipeptide antibodies for the various 7 subunits is best demonstrated by immunoblotting proteins of the appropriate sizes in cholate extracts from Sf9 cells (Spodopterafrugiperda ovary) infected with recombinant baculoviruses encoding the various 7 subunits)'~3 As shown in Fig. 3, the cholate-soluble membrane extracts from Sf9 cells infected with recombinant baculoviruses encoding the 71,72, 73,75, and 77 subunits were resolved on a 15% polyacrylamide-SDS gel, transferred to nitrocellulose, and then immunoblotted. As expected, each of the antibodies generated against a peptide based on sequence unique to the 71, 72, 73, 75, and 77 subunits recognized a protein of the appropriate size (5-7 kDa range) only in the cholate-soluble membrane extract from cells infected with the virus for the corresponding 7 subunit. The specificity of each of these antibodies was further demonstrated by the failure of each of these antibodies to detect similarly sized proteins in extracts from cells infected with viruses for the noncorresponding 7 subunits, or with the wild-type virus. 13j. D. Robishaw, V. K. Kalman, and K. L. Proulx, Biochem. J. 286, 672 (1992).
506
G/3y SUBUNITS
[39]
Usefulness of Antibodies
With the production of antibodies to the different y subtypes, it is now possible to determine the distribution of the y subtypes within various tissues, to examine the localization of the y subtypes within particular cell types, and to follow the synthesis, posttranslational processing, and assembly of particular a,/3, and y subunits of the G proteins. This information is ultimately needed to determine the role of specific combinations of a/3y subunits of the G proteins in a large number of different receptormediated signaling pathways. Tissue Distribution and Localization o f y Subunits The identification of the 3' subunits in a tissue or cell type is greatly facilitated by the use of subtype-specific antibodies) Such studies have lagged behind those with the a and/3 subunits owing to the inability of antibodies against the y subunits to detect these proteins in whole cell or membrane extracts by immunoblotting. As discussed above, by incorporating a high-temperature transfer step to allow the antipeptide antibodies to gain access to the epitopes, it has become possible for the first time to utilize these antibodies to determine the tissue distribution of the y subunits. The preparation and solubilization of membranes from several bovine tissues with 0.9% cholate have been described previously) In contrast to the/3 subtypes, the various y subtypes showed a more selective pattern of expression. Thus, the Yz and Y3 subunits were preferentially expressed in brain, whereas the Y5 and "Y7subunits were widely expressed in brain, heart, kidney, spleen, liver, and lung (Fig. 4). The Yl subunit was exclusively expressed in the retina (data not shown). This pattern of distribution for the 3' subunits is reminiscent of that for the o~ subunits, that is, if not found largely in the brain or retina, the a subunits were found in a variety of tissues. Ascertainment of the tissue distribution of specific subunits will be of importance in determining which of the large number of possible a/3y combinatorial associations actually occur in a physiological setting. The subtype-specific antibodies have also been successfully used for the cellular localization of the y subunits in monkey retina, 14 which contains readily distinguishable cell types with highly specialized functions. The/31 and Yl subunits were found to be localized in the rod outer segments (ROS), whereas the/33 and "Y2subunits were found to be localized in the cone outer segments. It is likely that the localization of particular a as 14 y . W. Peng, J. D. Robishaw, M. A. Levine, and K. W. Yau, Proc. Natl. Acad. Sci. U.S.A. 89, 10882 (1992).
[39]
ANTIBODIES
AGAINST
G-PROTEIN
'y S U B U N I T S
507
"o t__ "10
~
.t,-.
m
tl~
Idea
6.5~
%
6.5 m
3's
e.5
3'a
6"5m
'~'2 1
2
3
4
5
6
7
FIG. 4. Tissue distribution of y subunits. Cholate extracts of particulate fraction~ from the indicated tissues (100/.tg) were subjected to SDS-PAGE and immunoblot analysis, as described in the text. The standard shown in lane l represents 4/~g of purified bovine brain G proteins. The 6.5-kDa molecular mass marker was aprotinin. Exposure times were 2 and 4 days for the Y2and "/3 blots, respectively, and 18 hr for the Y5and ')17blots.
well as fly subunits within different cell types in the retina reflects the association of these subunits into different G proteins, with the phototransduction properties characteristic of the rod and cone outer segments. A similar a p p r o a c h to colocalize a , / 3 , and Y subunits in specific cell types in other tissues m a y provide a useful w a y of defining the n u m b e r o f possible a[3y associations that can o c c u r in a physiological setting. Processing o f y Subunits
The subtype-specific antibodies have also b e e n used to examine the posttranslational processing of the y subunits, which first involves the a t t a c h m e n t of a prenyl group to a cysteine residue near the carboxyl terminus of these proteins. 15,16 Insect (Sf9) cells expressing the Y2 and 3'3 subunits were fractionated into cytosolic and particulate fractions. Following extraction with 0.9% cholate, the particulate fractions were then frac15w. A. Maltese and J. D. Robishaw, J. Biol. Chem. 265, 18071 (1990). 16S. M. Mumby, P. J. Casey, A. G. Gilman, S. Gutowski, and P. C. Sternweis, Proc. Natl. Acad. Sci. U.S.A. 87, 5873 (1990).
508
Gfl~/ SUBUNITS
[39]
tionated into cholate-soluble and cholate-insoluble particulate fractions. As shown in Fig. 5, the nonprenylated forms of the 3,2 and 3,3 subunits, which migrated m o r e quickly on the gel, 13 were expressed largely in the cytosolic and insoluble particulate fractions, respectively (compare u p p e r and lower portions of Fig. 5). Although the reason for the difference in subcellular localization is not known, the localization of the 3'3 subunit in the insoluble particulate fraction m a y reflect an increased tendency of the 3'3 subunit to aggregate on overexpression. Conversely, the prenylated forms of the 3"2 and 3'3 subunits, which migrated m o r e slowly, 13 were e x p r e s s e d exclusively in the soluble and insoluble particulate fractions. The origin of a third f o r m of the 3'3 subunit in the insoluble particulate fraction has not b e e n determined, but it appears to represent an aggregated f o r m of the 3"3 subunit. Nevertheless, the localization of the prenylated
3'=-Infected
3'=,8=-Infected kDa
:Da
%-Infected
%~6~-Infected
%~=-Infected kDa
:Da
FIG. 5. Expression and subcellular distribution of various combinations of the ill, t2, ~t2, and )t3 subunits of the G proteins in Sf9 cells. At 72 hr after infection with various combinations of viruses encoding the ill, BE, 3t2, and 73 subunits, cells were fractionated to yield the cytosolic (C), cholate-soluble particulate (SP), and cholate-insoluble particulate
(IP) fractions. The same percentage 05%) of each fraction was resolved by SDS-PAGE on a 15% polyacrylamide gel, then transferred to nitrocellulose blots. The nitrocellulose blots were incubated with either the 72 antibody B-17 (top) or the 73 antibody B-53 (bottom), using ~2S-labeledgoat anti-rabbit secondary antibody for detection. The blots were exposed to film for 2 days with an intensifying screen.
[40]
ISOPRENYLATION
OF
G3,
AND G-PROTEIN
EFFECTORS
509
forms of the 3'2 and 3'3 subunits in the soluble particulate fraction is consistent with the role of prenylation in promoting membrane interaction. 17,18 Interestingly, the proportion of the prenylated forms of the 3'2 and 3'3 subunits was significantly increased by coexpression of the fll or/32 subunits in insect cells, 13 as demonstrated by an increase in the amount of the slower migrating forms in the soluble particulate fractions from/33'infected cells compared to 3,-infected cells (Fig. 5). Although the mechanism of this effect is not known, association of the/3 and 3' subunits into a functional heterodimer does not appear to be required for two reasons. First, the 3, subunit is able to undergo prenylation when expressed alone. Second, both the/31 and/32 subunits are able to enhance prenylation of the 3'3 subunit even though a number of investigators have shown that the /32 and 3"3 subunits do not associate to form a functional dimer. 19-2~ Acknowledgments This work was supported by National Institutes of Health Grant GM 39867 and an American Heart Association Established Investigatorship to J.D.R.
17 W. F. Simonds, J. E. Butrynski, N. Gautam, C. G. Unson, and A. M. Spiegel, J. Biol. Chem. 266, 5363 (1991). ta K. H. Muntz, P. C. Sternweis, A. G. Gilman, and S. M. Mumby, Mol. Biol. Cell3, 49 (1992). 19j. Iniguez-Lluhi, M. Simon, J. D. Robishaw, and A. G. Gilman, J. Biol. Chem. 7,67, 23409 (1992). 20 C. J. Schmidt, T. C. Thomas, M. A. Levine, and E. J. Neer, J. Biol. Chem. 1,67, 13807 (1992). 21 A. N. Pronin and N. Gautam, Proc. Natl. Acad. Sci. U.S.A. 89, 6220 (1992).
[40] I s o p r e n y l a t i o n o f y S u b u n i t s a n d G - P r o t e i n E f f e c t o r s
By
BERNARD K . - K . F U N G , JANMEET S. A N A N T , W U N - C H E N L I N , OLIVIA C. O N G , a n d HARVEY K . YAMANE
Introduction
Protein isoprenylation is a recently discovered posttranslational modification.1 The covalent addition of a isoprenyl moiety to a polypeptide was first described in the yeast mating factors. 2 These short polypeptides, I S. Clarke, Annu. Rev. Biochem. 61, 355 (1992). 2 y. Kamiya, A. Sakurai, S. Tamura, and N. Takahashi, Biochem. Biophys. Res. Commun. 83, 1077 (1973).
METHODS IN ENZYMOLOGY, VOL. 237
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.