the of bioloc~cal chemistry vol. no. 21, july pp. ic inc ... · toxin-mediated adp-ribosylation of...

THE JOURNAL OF BIOLOC~CAL CHEMISTRY I C 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 21, Issue of July 25, pp. 14072-14081, 1991 Printed in U.S.A.

Structural Analysis of Rod GTP-binding Protein, Gt LIMITED PROTEOLYTIC DIGESTION PATTERN OF G, WITH FOUR PROTEASES DEFINES MONOCLONAL ANTIBODY EPITOPE*

(Received for publication, June 25, 1990, and in revised form, November 9, 1990)

Maria R. Mazzoni, Justine A. Malinski, and Heidi E. HammS From the Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60680

The epitope of monoclonal antibody (mAb 4A), which recognizes the a subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chern. 262, 10839-10847) and the amino terminus (Navon, S . E., and Fung, B. K.-K. (1988) J. Biol. Chern. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on at and to resolve the discrep- ancy between these results limited proteolytic digestion of G, or at using four proteases with different substrate specificities has been performed. Endopro- teinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the ma- jority of at into two fragments of 34 and 5 kDa. The at 34-kDa fragment in the holoprotein, but not wguan- osine 5’-0-(3-thiotriphosphate), wasconverted further to a 23-kDa fragment. A small fraction of at-GDP was cleaved into 23- and 15-kDa fragments. Endoprotei- nase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 2 1 amino acid residues from the amino-terminal region of at, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved at progressively into fragments of known amino acid sequences (38, then 32 and 5, then 2 1 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met’ to ArgZo4. Thus the main portion of the mAb 4A antiqenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for rec- ognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments

* A preliminary report of this work was presented at the Associa- tion for Research in Vision and Opthalmology Meeting, Sarasota, FL, April 29-May 4, 1990. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ T o whom correspondence should be sent: Dept. of Physiology and Biophysics, University of Illinois College of Medicine, Box 6998, Chicago, IL 60680. Tel.: 312-996-7151.

containing the carboxyl terminus, showing that the amino-terminal residues from Met’ to Lys” were es- sential for antibody binding to at.

The light activation of rhodopsin, the rod outer segment light receptor, triggers the activation of an enzyme cascade which leads to cGMP hydrolysis and membrane hyperpolari- zation (Liebman et al. 1987). A guanine nucleotide-binding regulatory protein called transducin or G,’ communicates the signal from light-activated rhodopsin to a cGMP phosphodiesterase (for review see Stryer and Bourne, 1986; Gilman, 1987; Hurley, 1987; and Liebman et al., 1987). Like all heter- otrimeric G proteins, G, can be resolved into two functional subunits, cyt (39 kDa) and /?rt (Pt, 36 kDa; yt, 8 kDa). Photo- isomerized rhodopsin catalyzes the exchange of GTP for GDP on cyt (Fung and Stryer, 1980; Fung et al., 1981), which leads to the dissociation of subunits and activation of G,. In its GTP-bound form, the cy, subunit activates the cGMP phosphodiesterase, and this activation is terminated when the bound GTP is hydrolyzed to GDP by an intrinsic GTPase activity (Wheeler and Bitensky, 1977). Although the Pyt subunit does not participate directly in either GTP hydrolysis or phosphodiesterase activation its presence is important for effective binding of at to light-activated rhodopsin and GTP- GDP exchange (Fung, 1983).

The key role of G proteins in signal transduction has evoked much interest in their structure and function and in the mechanism of their activation by receptors (for a review, see Gilman, (1987)). G, binds tightly to light-activated rhodopsin in intact rod outer segment membranes (Kuhn, 1980). I t also binds selectively to unbleached rhodopsin in rod outer segment membranes (Hamm et al., 1987) and in reconstituted phospholipid vesicles (Fung, 1983). The cy, subunit can be ADP-ribosylated either by cholera toxin or pertussis toxin. Pertussis toxin, which catalyzes the transfer of ADP-ribose to CYS:’~~ (West et al., 1985), prefers the a,-GDP-bound form in the G, complex as substrate (Van Dop et al., 1984). The carboxyl-terminal region of at takes part in rhodopsin binding (Hamm et al., 1988) just as this region of cys is important for interaction with the P-adrenergic receptor (Sullivan et al., 1987). Pertussis toxin ADP-ribosylation can uncouple pertussis toxin substrates, G, , Gi, and Go from their cognate recep-

I The abbreviations used are: G,, photoreceptor guanyl nucleotide- binding protein; at, the a subunit of G,; pyt, the 0 and y subunit of G,; ROS, rod outer segment; mAb, monoclonal antibody; GTPyS, guanosine 5’-0-(3-thiotriphosphate); SDS, sodium dodecyl sulfate; MOPS, 3-(N-morpholino)propanesulfonic acid; DTT, dithiothreitol; PMSF, phenylmethanesulfonyl fluoride; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride; TPCK, L-l-tosylamido-2- phenylethyl chloromethyl ketone; ELISA, enzyme-linked immuno- sorbent assay; HPLC, high pressure liquid chromatography.

14072

mAb 4A Epitope Mapping of G, 14073

tors (Kurose et al., 1983; Cote et al., 1984). Additionally the amino-terminal region of a has been shown to be important for interaction with the @y subunit (Navon and Fung, 1987; Neer et al., 1988; Osawa et al., 1990b).

Specific antibodies against the a or /3 subunits of G proteins have been used widely to study the functional domains of these GTP-binding proteins (Cerione et al., 1988; McKenzie et al., 1988; Corrbze et al., 1987; Navon and Fung, 1988; Simonds et al., 1989). Monoclonal antibody 4A, which is directed against the a subunit of frog G,, also recognizes the a subunit of bovine Gt (Witt et al., 1984) and cross-reacts with the a subunits of Gi and G, (at > ai > a,) (Yatani et al., 1988; Hamm et al., 1989). Binding of mAb 4A to G, blocks light activation of photoreceptor cGMP phosphodiesterase by disrupting Gt-rhodopsin interaction (Hamm et al., 1987), and the epitope is not available when Gt is bound to light-activated rhodopsin (Hamm et al., 1987; Navon and Fung, 1988). The binding of mAb 4A to at also disrupts the G, complex, with dissociation of subunits (Navon and Fung, 1988; Mazzoni and Hamm, 1989). Moreover, the antibody inhibits the pertussis toxin-mediated ADP-ribosylation of at a t C Y S " ~ ~ (Deretic and Hamm, 1987; Mazzoni and Hamm, 1989).

Localization of the mAb 4A antigenic site is important for understanding of the basis of these multiple effects. However, conflicting experimental results have been reported (Deretic and Hamm, 1987; Navon and Fung, 1988), resulting in assignment of the mAb 4A epitope at either the carboxyl (Deretic and Hamm, 1987; Hamm et al., 1988) or the amino teminus (Navon and Fung, 1988).'

The purpose of the present work was to reexamine and expand the proteolytic mapping of G, with mAb 4A binding. This was accomplished by cleaving either G, or a, with four different proteases and analyzing antibody binding to the fragments on Western blots. The cleavage sites on at for two of these proteases, endoproteinase Arg-C and endoproteinase Lys-C, were also localized by determining the amino-terminal sequence of the proteolytic fragments. Monoclonal antibody 4A recognized proteolytic fragments of 34 and 23 kDa which contained the amino terminus of at and did not bind at after removal of 17-21 amino-terminal residues.

EXPERIMENTAL PROCEDURES

Materials and Miscellaneous Methods-TPCK-treated trypsin and soybean trypsin inhibitor were purchased from Worthington. GTP, GDP, GTPyS, endoproteinase Arg-C, endoproteinase Lys-C, and TLCK were products of Boehringer Mannheim. Staphylococcus au- r ew V8 protease, ["2P]NAD and "'1-protein A were from ICN Biomedicals. Pertussis toxin and its catalytic subunit, the A protomer, were purchased from List Biological Laboratories. S. aurew cell suspension was from Bethesda Research Laboratories. All other chemicals were from Sigma or Fisher. Protein concentrations were determined by the Coomassie Blue binding method (Bradford, 1976), using y-globulin as standard (Bio-Rad). SDS-polyacrylamide gel (12.5 and 16%) electrophoresis was carried out according to the method of Laemmli (1970). Amino acid sequence analysis was performed using a pulsed liquid phase protein Sequencer (Applied Biosystems Inc. model 477A).

Preparation of G , at, and Pyt Subunit-Bovine ROS membranes were prepared according to Papermaster and Dreyer (1974), with some modifications. Freshly collected retinas were suspended in a sucrose buffer solution containing 28% (w/w) sucrose, 60 mM KCl, 30 mM NaC1, 2 mM MgCl,, 1 mM DTT, 0.1 mM PMSF, and 10 mM MOPS (pH 7.5). The bovine retina suspension was gently stirred on ice and then centrifuged at 4,000 rpm (SS-34 rotor, Sorvall Instru- ments) at 4 "C for 4 min. Suspended ROS membranes contained in the supernatant were decanted while the pellet was resuspended in

' While this paper was under review a similar finding was reported (Hingorani, V. N., and Ho, Y.-K. (1990) J. Biol. Chen. 265, 19923- 19927).

fresh sucrose buffer solution and centrifuged a t 6,000 rpm (SS-34 rotor) at 4 "C for 4 min. The supernatant was decanted, and the pellet was discarded. The combined supernatants were diluted with buffer (60 mM KC1, 30 mM NaC1,2 mM MgClx, 1 mM DTT, 0.1 mM PMSF, and 10 mM MOPS, pH 7.5) and centrifuged at 19,000 rprn (SS-34 rotor) a t 4 "C for 20 min. The resulting pellet was resuspended in 24% (w/w) sucrose buffer solution and homogenized. The homogenized membranes were layered on the top of a discontinuous gradient containing 10 mM MOPS, pH 7.5, 60 mM KC1, 30 mM NaCl, 2 mM MgCl,, 1 mM DTT, and sucrose in incremental 8-ml density steps of 1.09, 1.11, and 1.13 g/ml. The gradient was centrifuged a t 25,000 rpm (SW 28 rotor, Beckman Instruments) at 4 "C for 30 min. The dense red band of ROS membranes which appeared at approximately the 1.09/1.11 g/ml interface was collected carefully. The ROS membranes were diluted with buffer and centrifuged at 19,000 rpm (SS-34 rotor) a t 4 "C for 20 min. The resulting pellet was resuspended in buffer, homogenized, and stored a t -70 "C. Bovine G, was extracted from ROS membranes as described by Stryer et al. (1983). The a,-GTPyS and firt subunits were extracted from ROS membranes by using GTPyS and then purified by chromatography on Blue-Sepharose CL- 6B (Pharmacia LKB Biotechnology Inc.), essentially as described by Kleuss et al. (1987). All the purified proteins were stored in 40% glycerol a t -20 "C.

Proteolysis of G, and at Subunit-Trypsinization of Gt and a,- GTPyS was performed essentially as described by Fung and Nash (1983). Either G, (2 mg/ml) or o(,-GTPyS (1 mg/ml) in buffer A (200 mM NaC1,2 mM MgCl,, 1 mM DTT, 10 mM MOPS, pH 7.5) containing 25% glycerol was incubated with an equal volume of TPCK-treated trypsin (0.08 mg/ml) a t 0 "C. At the indicated times the reactions were terminated by incubating an aliquot of the reaction mixtures with trypsin inhibitor (trypsin/trypsin inhibitor ratio, 1:lO (w/w)). The samples, after adding the electrophoresis sample buffer (Laem- mli, 1970), were boiled for 5 min.

Proteolysis of G, and a,-GTPyS by S. aurew V8 protease was carried out as described by Navon and Fung (1987). Either GI (1.2 mg/ml) or a,-GTPyS (0.6 mg/ml) was digested in buffer A by S. aureus V8 protease for 2 h a t room temperature. The protease to a,- GTPyS ratio was 1:50 (w/w). The reactions were stopped by the addition of TLCK and PMSF to a final concentration of 2.4 and 2.3 mg/ml, respectively. Then, unless stated otherwise, the electrophoresis sample buffer (Laemmli, 1970) was added, and the samples were boiled for 5 min.

Proteolysis of G, and a,-GTPyS by either endoproteinase Arg-C or endoproteinase Lys-C was carried out in buffer A a t room temperature. For endoproteinase Arg-C, either G, (1.6 mg/ml) or a,-GTPyS (0.8 mg/ml) was digested at a protease/(=, ratio of 1:3 (w/w). At each time point, aliquots of the reaction mixtures were removed, and the reactions were stopped by the addition of TLCK to a final concentration of 2.4 mg/ml. The samples, after addition of the electrophoresis sample buffer (Laemmli, 1970), were then heated a t 100 "C for 5 min. Endoproteinase Lys-C was used to cleave either G, (1 mg/ml) or at- GTPyS (0.5 mg/ml) at a protease/(=, ratio of 1:lOO (w/w). Aliquots of the reaction mixtures were removed a t different times, and proteolysis was terminated by the addition of TLCK at a final concentration of 54 pg/ml. The electrophoresis sample buffer (Laemmli, 1970) was added, and the samples were boiled for 5 min. When the proteolytic fragments were purified by reversed phase HPLC (Brownlee, Aquapore RP-300, C8) the reactions were stopped by the addition of 1% trifluoroacetic acid and 6 M guanidine HCl (final concentrations), applied to the column, and eluted with a linear acetonitrile gradient in 0.1% trifluoroacetic acid.

ADP-ribosylation of G, and a,-GTPyS-Pertussis toxin-catalyzed ADP-ribosylation of either G, or a,-GTPyS was carried out as described previously (Mazzoni and Hamm, 1989). The ['"PIADP-ribosylated proteins were substrates for proteolysis by trypsin, endoproteinase Arg-C, and endoproteinase Lys-C.

["'PIADP-ribosylation of Gt prior to sequencing was catalyzed by the active subunit alone, the A protomer, a t a ratio of 50:l (G,/A protomer, w/w) for 90 min at 30 "C. Labeled G, was separated from the A protomer and unreacted [:'2P]NAD by gel filtration on Sephacryl S-300 equilibrated in buffer A followed by repeated washing with buffer A of the pooled protein in a Centricon 30 filtration unit (Amicon).

Immunological Procedures-Monoclonal antibody 4A was gener- ated by immunization of mice with the soluble fraction of ROS proteins, purified, and characterized as described by Hamm and Bownds (1984) and Witt et a/. (1984). The proteins were transferred from the SDS-polyacrylamide gel to nitrocellulose (0.1 pm, Schleicher

14074 mAb 4A Epitope Mapping of Gt & Schuell) essentially as described by Towbin et al. (1979). The immunoblots were incubated in 150 mM NaCl, 50 mM Tris/HCl, pH 8.5 (TBS), containing 3% ovalbumin (OTBS) for 3 h at room temperature to block nonspecific binding and then transferred to OTBS containing mAb 4A (50 pg/ml) and incubated overnight. After two washes in TBS, one wash in TBS containing 0.1% Nonidet P-40 followed by an additional wash in TBS the immunoblots were incubated in OTBS for 3 h and then transferred to OTBS containing 'T- protein A (0.5 pCi/ml, specific activity, 30 pCi/pg) and incubated at room temperature. After 4 h of incubation the immunoblots were rinsed as described above and dried. Autoradiography was performed by exposing the immunoblot to a Kodak XAR-2 x-ray film with an intensifying screen. Immunoprecipitation of at and at after cleavage by S. aureus V8 protease was carried out as described previously (Mazzoni and Hamm, 1989). After digestion of at by the protease, the sample was treated with TPCK, TLCK, and PMSF at final concentration of 5 mM each, and the pH was adjusted to pH 7.5 before the addition of mAb 4A. The antigen-antibody immunocomplexes were precipitated using S. aureus cell suspension as described (Mazzoni and Hamm, 1989).

RESULTS

To determine the localization of mAb 4A epitope on the at subunit, antibody binding to proteolytic fragments of a, was analyzed. Four different proteases were used to cleave either G, or the at subunit. The fragments were separated by SDS- gel electrophoresis (Laemmli, 1970), transferred to nitrocellulose, and mAb 4A binding to these fragments was determined using lZ51-protein A.

Trypsin was the first protease used to cleave either G, or at since the proteolytic fragments are well characterized (Fung and Nash, 1983; Hurley et al., 1984). The time course of limited tryptic digestion of G, on a 16% SDS-polyacrylamide gel is shown in Fig. 1A (left panel). Transient proteolytic fragments (a38, a34, and a3J appeared; however, the major final fragments were aZ1, a12, a5, p 2 3 , and p14, in agreement with Fung and Nash (1983) and Hurley et al. (1984). A duplicate gel was blotted onto nitrocellulose, and immunoblot analysis showed that mAb 4A bound to the a, subunit and two transient fragments of 34 and 23 kDa (Fig. lA, right panel). The 23-kDa fragment could not be localized in the gel since it comigrated with and was obscured by the more abun- dant f l 23 fragment. The cleavage sites on at with the sequential formation of the 38-, 32-, 5-, 21-, and 12-kDa fragments are known (Hurley et al., 1984). However, the origin of the 34- and 23-kDa fragments has not been elucidated clearly (Fung and Nash, 1983).

To determine if the 34- and 23-kDa fragments still contained the carboxyl-terminal region of at trypsinization was carried out using G, previously 32P-labeled at Cys347 by pertussis toxin-mediated ADP-ribosylation (Fig. 2). The quantity of the 34-kDa fragment was reducedfor both ADP-ribosylated protein and G, digested with trypsin under conditions used for ADP-ribosylation which included increased reducing agent. However, the overall pattern of tryptic peptides was the same for both unmodified and ADP-ribosylated Gt i.e. the same major final fragments ( a z l , a12, as, p 2 3 , and Addi- tionally, the overall conformation of the ADP-ribosylated Gt used in the experiments was very similar to the native conformation of the protein since studies of various pertussis toxin substrates have shown that the effect of ADP-ribosylation is at the level of receptor-&-protein interaction. ADP- ribosylated Gi and Go show normal nucleotide binding and GTPase activity (Vi, 1986; Sunyer et al., 1989) as well as unchanged interaction with Py (Neer and Clapham, 1990). Similiarly, for G,, Ramdas and Wensel (1990) have shown that although pertussis toxin labeling blocks rhodopsin-catalyzed nucleotide exchange there is no effect on the rate of nucleotide exchange in the absence of light-activated rhodop-

sin and that modified Gt also activates photoreceptor cGMP phosphodiesterase in the presence of AlF4-.

The autoradiogram (Fig. 2, right panel) shows that at, a38, and as are 32P-labeled as well as an early minor fragment (15 kDa) seen in the Coomassie Blue-stained gel at 1, 2, and 5 min. This transient fragment, the product of cleavage at rather than at Ar$'', has also been reported by Fung and Nash (1983). The 34- and 23-kDa fragments were not 32P- labeled and therefore had lost the carboxyl-terminal region containing Cys347.

When a,-GTPyS was used as substrate for trypsinization the cleavage pattern was different from that of G, (Fig. 1, compare A and B, left panels). The amount of the 34-kDa fragment was decreased dramatically, and the 32-kDa fragment was not cleaved further to 21- and 12-kDa fragments. Accumulation of the 34-kDa fragment appeared to be depend- ent on the conformation of the a, subunit, the presence of reducing agents (compare Fig. lA and Fig. 2) and the Byt subunit (compare Fig. 1, A and B). For a,-GTPyS the absence of Pyt and the presence of GTPyS enhanced cleavage at Lys" and protected a t Are4 . The two protein bands of 23 and 14 kDa represent trypsin and a trypsin fragment resulting from enzyme self-digestion, respectively. Western blot analysis of mAb 4A binding to the fragments (Fig. lB, right panel) showed that the antibody bound only the at subunit and the 34-kDa fragment.

S. aureus V8 protease removes 21 residues from the amino terminus of at (Navon and Fung, 1987), with the formation of a stable 38-kDa fragment. Using Western blot analysis, Navon and Fung (1988) have shown that mAb 4A does not recognize the 38-kDa fragment. In a similar experiment (Fig. 3), a,-GTPyS was cleaved with S. aureus V8 protease. After blocking the reaction, aliquots containing 20 pg of protein were applied to two gels for protein stain and immunoblot, respectively. Immunoblot analysis (Fig. 3, rightpanel) showed that mAb 4A did not bind to the 38-kDa fragment, which lacked the amino-terminal 21 amino acids. In addition, mAb 4A did not immunoprecipitate the 38-kDa fragment (Fig. 4, lane 4 ) but was capable of immunoprecipitating uncleaved a, (Fig. 4, lane 2 ) . Control experiments showed that the presence of the enzyme and inhibitors did not affect the immunoprecipitation of uncleaved at (data not shown).

As a further step in the analysis of mAb 4A binding to proteolytic fragments of at, the cleavage pattern of either G, or a, after digestion with endoproteinase Arg-C was determined. Ho et al. (1989) reported that endoproteinase Arg-C makes a single cut at Arg3I0 of at, generating a 34- and a 5- kDa fragment containing the amino and carboxyl termini of at, respectively. Fig. 5A shows the time course of ADP- ribosylated G, obtained by digestion with endoproteinase Arg- C. The Coomassie Blue-stained gel (Fig. 5A, leftpanel) shows that although at was not a particularly good substrate for this enzyme, digestion resulted in 34- and 23-kDa fragments, and the proteolytic pattern was the same whether Gt or ADP- ribosylated G, was used as substrate (Fig. 5, compare A and B left panels). The fl, and y, subunits were uncleaved. In the autoradiogram (Fig. 5A, right panel), the 32P label was associated mainly with the at subunit, a 5-kDa fragment, and a minor -15-kDa fragment. The 34- and 23-kDa fragments were not 32P-labeled, indicating that the fragments did not contain Cys347. Immunoblot analysis of these proteolytic fragments showed that the antibody bound a,, the 34- and 23- kDa fragments, and a -12-kDa fragment which appeared very late in the proteolysis (Fig. 5B, right panel). The total inten- sity of the '2sI-labeled bands in each lane appeared to be

mAb 4A Epitope Mapping of G, A

Coomassie blue Stain

Mrx 1 0-3 - 66-

45- 36- ‘ 24- 29-

20- - 14-

0 G.1112 1 2 5 10 15 30 4 5 6 0 90 1 2 0 1 8 0

time (mid

Coomassie blue slain M ~ ~ ~ o - ~

66-

45- 36- 29- 24-

-cud “ a 3 4

-61 4 - a 1 2

”Y

-tu5

mAb 4A Western-Blot

”- “-

G 4 112 1 2 5 10 15 30 4 5 6 0 9 0 1 2 0 180

time ( m i d

mAb 4A Western-Blot

-T

14075

-11 “134

‘“23

20-

14- T

FIG. 1. Time course of proteolysis of G, and at-GTPyS by TPCK-treated trypsin and Western blot analysis of mAb 4A binding to the fragments. Purified bovine G, (1 mg) or a,-GTPyS (0.5 mg) was digested with 40 pg of TPCK-treated trypsin in 1 ml of buffer A (see “Experimental Procedures”) on ice. At the indicated time points, 80 pl of the mixture was removed, and the reaction was stopped by adding 4 pl of trypsin inhibitor (8 mg/ml). The proteolytic fragments (A, 35 pg of Gtllnnc: B, 17 pg of n,/lane) were separated by electrophoresis on duplicate SDS-polyacrylamide gels (16%). One gel was stained with Coomassie Blue ( A and B, left panels), and the other ( A and B, right panels) was blotted onto nitrocellulose. Antibody binding to fragments was detected as described under “Experimental Procedures.” Molecular weight standards are indicated, as are the sizes and origin of the tryptic fragments. C, G,; N, a,-GTPyS; T, trypsin; I, trypsin inhibitor.

equal, suggesting that the at subunit and its fragments bound a similar amount of antibody.

When compared with G,, the cleavage pattern of a,-GTPyS showed that the 23-kDa fragment was not produced (Fig. 6). This suggests that the presence of GTPyS prevented endoproteinase Arg-C cleavage at Argn4, similar to digestion with trypsin, in which bound GTPyS eliminates A r e ’ as a cleavage site (Fig. 1B) (Fung and Nash, 1983). A fraction of the 34-kDa fragment was converted to two fragments of -33 and 32 kDa after many hours of digestion (Fig. 6).

To define both cleavage sites on at the fragments obtained after digestion of [3’P]ADP-ribosylated GI were purified by

reversed phase HPLC and sequenced. The analysis revealed that the 5-kDa fragment had an amino-terminal sequence of Asp-Val-Lys-Glu-Ile-Tyr-Ser-His-Met-Thr-Cys-Ala-Thr- Asp, and the 15-kDa fragment had an amino-terminal sequence of Lys-Lys-Trp-Ile-His-Cys-Phe-Glu-Gly-Val-Thr- Cys-Ile-Ile, which corresponded to at residues 311-324 and 205-218, respectively (Yatsunami and Khorana, 1985; Tanabe et al., 1985; Lochrie et al., 1985; Medynski et al., 1985). We were unable to sequence the 34- and 23-kDa fragments. This suggested that the 34-kDa fragment contained the amino terminus which is known to be blocked to sequencing (Hurley et al., 1984). Therefore, in agreement with a previous report

14076 mAb 4A Epitope Mapping of G,

-3 Coomassie blue stain Mr x 10

66-

45- 36- "" .- -

PT 32P-ADP Ribosylated G

/ a 38

G + l 1 2 5 15 30 60 180 G + I 1 2 5 15 30 60 180

time (min) time ( m i d FIG. 2. Time course of proteolysis of pertussis toxin ADP-ribosylated G, by TPCK-treated trypsin.

Purified bovine GI (325 pg) was ADP-ribosylated with pertussis toxin (PT) as described (Mazzoni and Hamm, 1989) and digested immediately with 13 pg of TPCK-treated trypsin in 380 p1 of reaction mixture. The reaction was stopped by adding 2 pl of trypsin inhibitor (8 mg/ml) to 47 pl of the reaction mixture at the time points indicated. The fragments (35 pgllane) were separated by electrophoresis on an SDS-polyacrylamide gel (16%). The gel was stained with Coomassie Blue (left panel), dried, and autoradiographed (right panel) using a Kodak XAR-2 film with an intensifying screen. Molecular weight standards and tryptic fragments are labeled as described in Fig. 1.

Mr x "

97-

66-

45-

24-

20-

14-

a- a- 38

a

1 2 1 2

FIG. 3. Digestion of at-GTPyS by S. aureus V8 protease and Western blot analysis of mAb 4A binding to the fragment. Purified n,-GTPyS (40 pg) was digested with 0.8 pg of S. aureus V 8 protease in 58 pl of buffer A for 2 h a t room temperature. The reaction was stopped by adding 5 pl of PMSF (35 mg/ml) and 10 pl of TLCK (18 mg/ml). The sample (20 pg of protein/lane) was loaded on duplicate 12.5% SDS-polyacrylamide gels and electrophoresed. One gel was stained with Coomassie Blue (left panel), and the other (right panel) was blotted onto nitrocellulose. Antibody binding to the 38- kDa fragment was detected as described under "Experimental Pro- cedures.'' lane I, n,; lane 2, aR8.

- -HC

--LC

1 2 3 4

FIG. 4. Immunoprecipitation of a, and a, after digestion with S. aureus VS protease (axR) by mAb 4A. Purified a,-GTPyS (10 kg) was digested with 0.2 pg of S. aurew V8 protease in 21 pI of buffer A containing 0.5 mM DTT for 2 h a t room temperature. The reaction was stopped by the addition of 2 p1 of TPCK (50 mM), 2 pI of TLCK (50 mM), and 1 pl of PMSF (100 mM). The sample volume was brought up to 100 pl with buffer A containing 0.4% Lubrol PX and 0.1 mM DTT, and the pH was adjusted to 7.5. Monoclonal antibody 4A (76 pg) was added to the mixture and incubated for 1 h a t room temperature. The immune complex was precipitated as described previously (Mazzoni and Hamm, 1989). The immunopre- cipitated proteins were analyzed by SDS-polyacrylamide gel (12.5%) electrophoresis and stained with Coomassie Blue. Control, at (lane I); a, + mAb 4A (lane 2) ; control, nR8 (lane 3 ) ; 0 3 8 + mAb4A (lane 4 ) . HC and LC are antibody heavy and light chain, respectively.


FIG. 5. Time course of proteolysis of G , and pertussis toxin-catalyzed ADP-ribosylated G , by endoproteinase Arg-C and Western blot analysis of mAb 4A binding to fragments. A, purified G, (410 pg) was ADP-ribosylated with pertussis toxin ( P T ) as described previously (Mazzoni and Hamm, 1989) and then digested with 150 pg of endoproteinase Arg-C in 340 pl of reaction mixture a t room temperature. The reaction was stopped by adding 7.4 pl of TLCK (18 mg/ml) to 58 pI of the reaction mixture at the time points indicated. The proteolytic fragments (30 pg/lane) were separated on a 16% SDS-polyacrylamide gel. The gel was stained with Coo- massie Blue (left panel), dried, and autoradiographed (right panel). B , purified G, (520 pg) was digested with 160 pg of endoproteinase Arg-C in 315 pl of buffer A a t room temperature. At the indicated time points, 49 pl of the mixture was

by the addition of 7.4 p1 of TLCK (18 removed, and the reaction was stopped

mg/ml). The proteolytic fragments (40 pg/lane) were separated on duplicate 16% SDS-polyacrylamide gels. One gel was stained with Coomassie Blue (left panel), and the other (right panel) was blotted onto nitrocellulose, and mAb 4A binding to the fragments was detected as described under “Experimental Proce- dures.” Molecular weight standards and proteolytic fragments are labeled as described in Fig. l. G, G,; I, TLCK; E, endoproteinase Arg-C.

A

Coomassie blue s t a m

~ ~ ~ 1 0 - 3 _ _

6 6 -

4 5 - 3 6 - 2 9 - 2 4 -

20-

14-

G 4 1 3 6 2 4 3 6 E G4 1 3 6 2 4 3 6 E

time (h) time (h)

B Coomassie blue stain

Mrx 1 0-3 ~~ ~ ~ ~~ ._- ”””

6 6

4 5 3 6

2 9 2 4 20

PT 32P-ADP Rlbosylated G

mAb 4A Western-Blot

14

-1

4 t 5

G G.1 1 3 6 1 2 2 4 3 6 E G G.1 1 3 6 12 24 36 E

flme (h) hme (h)

- ( X 5

- (r “Y34

“y23

(Ho et al., 1989), the first cleavage occurred at Arg3”. Addi- tionally, we observed a second cleavage a t Arg‘04.

The fourth protease used to cleave either G, or the at subunit was endoproteinase Lys-C. Fig. 7 shows the time course of proteolysis of pertussis toxin-catalyzed ADP-ribosylated G, ( A , left panel) and a,-GTPyS ( B , left panel). A similar cleavage pattern was obtained when either unmodified GI or a,-GTPyS was used as substrate for digestion with endoproteinase Lys-C (data not shown). The Coomassie Blue- stained gel of digested a,-GTPyS (Fig. 7B, left panel) shows that at was cleaved to a transient 38-kDa fragment, which was further converted to a stable 36-kDa fragment. At a protease/a, subunit ratio of 1:50 (w/w) the proteolysis was almost complete after 2 h. When G, was used as substrate (Fig. 7A, left panel), the 36-kDa fragment was not clearly visible since it was obscured by the PI band. The P, and y, subunits were not cleaved (data not shown). Autoradiography of the gels (Fig. 7 , A and B, right panels) shows that the :32P label remained associated with the 36-kDa fragment. When ADP-ribosylated G, was digested, a minor labeled fragment (-15 kDa) appeared late in the time course. These results suggested that the two major cleavage sites were located near

the at amino terminus. To determine the exact location of these cleavage sites on al-GTPyS the fragments were purified by reversed phase HPLC and sequenced. This resulted in assignment of amino-terminal sequences Lys-Leu-Lys-Glu- Asp-Ala-Glu-Lys-Asp-Ala and Asp-Ala-Arg-Val-Lys-Leu- Leu-Leu to the 38- and the 36-kDa fragments, respectively (a, residues 18-27 and 26-34) (Yatsunami and Khorana, 1985; Tanabe et al., 1985; Lochrie et al., 1985; Medynski et al., 1985). Therefore, endoproteinase Lys-C cleaves at first at Lys” and then at Lys”.

Western blot analysis of mAb 4A binding to the proteolytic fragments shows (Fig. 8, right panel) that the antibody recognized only ut and did not bind the major fragments, 38 and 36 kDa, which were missing the first 17 or 25 amino-terminal residues.

The major cleavage sites on at for the four proteases used in this work are summarized in Fig. 9. The figure clearly shows that at has three regions that are readily available for proteolytic digestion: Arc’.’, Arg””, and Lys”-Lys’”. Trypsin and endoproteinase Arg-C each had a common cleavage point a t Ar$04 and A&’”. Recent work with chymotrypsin:’ indi-

M. R. Mazzoni and Hamm, H.E., submitted for puhlication.

14078 mAb 4A Epitope Mapping of G,

Coomassie blue stain

M~~ 1 o - ~

66-

45- 36- "------ -a!

"""Pp 29- 2 4 -

20-

14-

"x34 """- -0""

mm""

-a! 5

3 6 12 24 36 E

time (h)

FIG. 6. Time course of proteolysis of a,-GTPyS by endoproteinase Arg-C. Purified o,-GTPyS (260 pg) was digested with 160 pg of endoproteinase Arg-C in 315 pI of buffer A at room temperature. At the indicated time points, 49 p1 of the mixture was removed, and the reaction was blocked by adding 5.4 p1 of TLCK (18 mg/ml). The proteolytic fragments (19 pg/ml) were separated with 16% SDS- polyacrylamide gel electrophoresis. Molecular weight standards and proteolytic fragments are labeled as described in Fig. 1. (Y, a,-GTPyS; I, TLCK; E, endoproteinase Arg-C.

cates that this enzyme too has a cleavage site near Ar$'.' since it cleaves at Trp'"'. The third region, Lys"-LyS", appeared to be especially sensitive to proteolytic digestion. Within that short segment endoproteinase Lys-C had two cleavage sites, Lysl' and Lys'", and trypsin and S. aureus V8 protease each had one, Lys" and Glu", respectively. Chymotrypsin also cleaves a, at two sites (Leu"" and Leu")" in this susceptible region. This region is very hydrophilic, and its accessibility to a variety of proteases confirms that it is surface exposed.

DISCUSSION

The major finding of this study is that the main portion of the mAb 4A epitope on a, is located in the amino-terminal region of a,. This conclusion is based on reanalysis and expansion of previous peptide mapping studies and clearly emphasizes the importance of the amino-terminal region. Specifically, after endoproteinase Arg-C and trypsin digestion of a,, mAb 4A hound the 34- nntl 23-kDa fragment,$. Since the endoproteinase Arg-C fragments were not sequenceable it was assumed that these fragments contained the amino terminus, which is reported to be blocked to sequencing (Hurley et al., 1984). The 34- and the 23-kDa fragments lack 41 and 106 carboxyl-terminal residues, respectively. Furthermore, mAb 4A did not recognize the tryptic, endoproteinase Lys-C, or S. aureus V8 protease 38- or 36-kDa fragments lacking 17- 25 amino-terminal residues. Since the 23-kDa fragment was recognized by the antibody, residues from Lys"l5 to Phe"" were not required for antibody binding. In contrast, the amino-terminal residues from Met' to Lys" appear to be required for antibody-a, binding.

Previous studies from this laboratory have suggested that mAb 4A binds to the carboxyl-terminal region of at. Deretic

and Hamm (1987) reported the binding of mAb 4A to a, tryptic and chymotryptic fragments using epitope mapping on Western blots. The results showed that none of the major tryptic fragments bound the antibody, but a 34-kDa fragment and several minor transient fragments of 26, 23, 18, 16 and 12 kDa which comigrated with '"P-ADP ribosylated fragments did bind the antibody. Size analysis of these antigenic fragments suggested that the main portion of the mAb 4A epitope could be localized to the carboxyl-terminal region between

. However, these fragments were not sequenced so their origins could not be proven.

In the present work the antibody bound the 34- and 23- kDa tryptic fragments. The transient fragments of 26, 18, 16, and 12 kDa were not normally observed when trypsin activity was completely blocked by trypsin inhibitor before the addition of electrophoresis sample buffer. However, when trypsin inhibitor and electrophoresis sample buffer were added together before the addition of G,, trypsin activity was not completely blocked, and the minor fragments appeared (data not shown).

The results of Deretic and Hamm (1987) were supported by ELISA competition studies between synthetic at peptides and G, for mAb 4A binding (Hamm et al., 1988). The a,

binding whereas peptide AspX-Ala"l did not compete (Hamm et al., 1988). Another amino-terminal peptide, Met'-Ala"', also had no effect (Hamm et al., 1990). Since the amino terminus of a, is blocked, acetyl-l-23-NH2 was recently syn- thesized, and this peptide also did not compete with c ~ , - G T P r S for mAb 4A binding (data not shown). Thus, competition ELISA suggested that the carboxyl terminus may also be recognized by the antibody. Navon and Fung (1988) have reported that a synthetic a, peptide derived from the amino-terminal sequence, Cys-Tyr-Asp"-Ala"', competes with GI for antibody binding in a solid phase immunoassay. However, the addition of two amino acids not present in the native sequence and the requirement for 10-fold higher concentrations of this peptide as compared with carboxyl-terminal peptides (Hamm et al., 1988) make this result difficult to interpret. Another difficulty in the interpretation of these contrasting results arises from the indirect nature of the measurement. Peptides that compete for antibody binding to GI may do so directly, by binding to the antibody itself or to a site on G, which is recognized by the antibody, or allosteri- cally, by binding cy1 a t a site away from, but affecting, the epitope. I t is not possible to distinguish among these alter- natives with peptide competition ELISA.

Complete studies on the antigenicity of other proteins such as lysozyme have shown that neither Western blot nor peptide competition ELISA is adequate to specify a monoclonal antibody epitope completely, as finally determined by x-ray crynt.allography of tllc tlllligell-anti~)ody cnmplcx. I n fwL, lor the known protein/Fab crystal structures, the epitope is usu- ally formed by several regions of the protein, with approximately 15 amino acids taking part in binding to the antibody (Amit et al., 1986; Sheriff et al., 1987; for review see Davies et al., 1988; Laver et al., 1990; Janin and Chothia, 1990).

The work presented here shows that on Western blots the presence of the amino-terminal region is the main criterion for antibody-a, binding. However, the epitope on the native protein could be linear, continuous over a single length of amino acids, or discontinuous, composed of amino acids from disparate parts of the primary sequence which are brought together on the surface when the protein assumes its native conformation. If the mAb 4A epitope is linear it could be contained completely within the amino terminus, as proposed

Arg:?It) and Lys:iZl

peptides Glu:'ll-Val:V28 and Ile?~lll-Phe:~~?ll inhibited antibody

FIG. 7 . Time course of proteolysis of pertussis toxin-ADP-ribosylated GI and cr,-GTPrS by endoproteinase Lys-C. Either G, ( A , 150 pg) or at- GTPyS ( R , 150 pg) was ADP-ribosylated with pertussis toxin ( P T ) as described (Mazzoni and Hamm, 1989) and then digested with endoproteinase Lys- C (1.5 and 3 pg, respectively) in 157 and 314 pI of the reaction mixtures, respectively. The reactions were blocked by adding 5 and 10 pl of TLCK (0.28 mg/ ml) to 21 and 42 pl of the reaction mixtures, respectively, at the time points indicated. The proteolytic fragments (20 pg/lane) were separated on 12.5% SDS- polyacrylamide gels. The gels were stained with Coomassie Blue ( A and R, left panels), dried, and autoradiographed ( A and R, right panels). E, endoproteinase Lys-C.


A 32 Coomassie blue stain PT P-ADP Rlbosylated G

Mrx 1 0-3

66-

45-

29- 24-

20- -" -CYl5

14- " rh - - - -. "y

G+l 1 15 30 60 120240 G + l 1 15 30 60 120240

time (mid time (mid

6 Coomassie blue stain

Mrx 10 -3

PT P-ADP Ribosylated 32

66-

14-

Q * l 1 1 5 3 0 6 0 1 2 0 2 4 0 a4 1 1 5 3 0 6 0 1 2 0 2 4 0

time (min) time (min)

by Navon and Fung (1988). However, Western blot analysis and peptide competition assays cannot determine if additional amino acids form a discontinuous epitope.

G, structure function studies have revealed that the rhodopsin binding domain is complex. The carboxyl terminus of f l I interacts directly with rhodopsin (Hamm et al., 1988,1991), but other regions of CY^ may also be involved in rhodopsin binding. Hingorani et al. (1988) suggested on the basis of cross-linking experiments that the rhodopsin binding domain of cy1 contains both the amino and carboxyl termini of the protein. We have found that amino-terminal peptides as well as carboxyl-terminal peptides can disrupt rhodopsin-G, inter-

action (Hamm et al., 1988), supporting this suggestion. How- ever, association of a1 and PT, subunits is required for G, interaction with rhodopsin, and the amino-terminal region of CY, has also been suggested as the site of interaction with P-y,. Proteolytic removal of the amino-terminal 23 amino acids from at causes dissociation from (Navon and Fung, 1987), and Gs/Gi chimera studies demonstrate that disruption of internal regions near the amino terminus (a , residues 15-41, 62-71, and 87-144) also inhibits interaction with PT (Osawa et al., 1990b). If rhodopsin binds both the amino and carboxyl termini then these regions may be physically close, as has been suggested by several recent studies (Dhanasekaran et al.,

14080

Mrx

66-

45 - 36- 29- 24-

20-

14-

mAb 4A Epitope Mapping of G,

Coomassie blue stain mAb 4A Western-Blot

I 1 5 30 60 120 240 360 E

time (min)

I 1 5 30 60 120 240 360 E

time (min)

FIG. 8. Time course of proteolysis of at-GTPyS by endoproteinase Lys-C and Western blot analysis of mAb 4 A binding to fragments. Purified n,-GTPyS (200 p g ) was digested with 4 pg of endoproteinase Lys-C in 440 pl of huffer A at room temperature. At the indicated time points, 50 p1 of the mixture was removed, and the reaction was stopped hy adding 5 pl of TLCK (0.52 mg/ml). The proteolytic fragments (10 pg/lane) were separated on duplicate 12.5!'E SDS-polyacrylamide gels. One gel was stained with Coomassie Blue (lrft panel), and the other was blotted onto nitrocellulose. mAh 4A binding to fragments was detected as described under "Experimental Procedures."

10 20 30 40 technical assistance, Dr. Paul Morris and the Protein Sequencing/ MGAGASAEEKHSRELEKKLKEDAEKDARTVKLLLLGAGES. . . . . . . . . . . . . . . . . . . . Synthesis Laboratory of the Research Resources Center at University

of Illinois at Chicago for the peptide sequencing, Dr. Yee-Kin Ho for suggesting the use of endoproteinase Arg-C, and Dr. A. Lucacchini for generous support.

t t t t t t C KTC V K

200 210 220 230 240 250 NFRMFDVGGQRSERKKWIHCFEGVTCIIFIAALSAYDMVLHESLHLFNSIC

t t R C T

260 270 280 290 300 310 NHRYFATTSIVLFINKKDVFSEKIKI(AHLSICFPDYNGPNTYEDAGNY1KVQFLEL~R

R T

t

320 330 340 DVKEIYSHMTCATDTQNVKFVFDAVTDIIIKENLKDCGLF

350

FIG. 9. Summary of the major proteolytic cleavage sites on a,. T, trypsin; K , endoproteinase Lys-C; V , Staphylococcus aurrus V8 protease; R , endoproteinase Arg-C; C, chymotr.ypsin; *, Cys'"', site of ADP rihosylation by pertussis toxin.

1988; Hingorani and Ho, 1988; Osawa et al., 1990a, 1990b). Monoclonal antibody 4A binding to GI has several impor-

tant functional consequences. It disrupts G, interaction with rhodopsin (Hamm et al., 1987) and causes G, subunit dissociation (Mazzoni and Hamm, 1989). G, binding to light- activated rhodopsin interferes with mAb 4A binding to its epitope (Hamm et al., 1987; Navon and Fung, 1988). The functional effects of mAb 4A are most likely related to the close functional relationship between the rhodopsin and By, binding sites on mI and may be suggestive of interaction between the amino and carboxyl termini of at.

Acknou*ledgments-We thank Lana Salov and Tony Garcia for

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the of bioloc~cal chemistry vol. no. 21, july pp. ic inc ... · toxin-mediated adp-ribosylation of...

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