©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Receptor and Membrane Interaction Sites on G
A RECEPTOR-DERIVED PEPTIDE BINDS TO THE CARBOXYL TERMINUS (*)

(Received for publication, July 13, 1995; and in revised form, December 14, 1995)

Joan M. Taylor (1) Gayatry G. Jacob-Mosier (2) Richard G. Lawton (2) Marcian VanDort (3) Richard R. Neubig (1) (4)(§)

From the  (1)Departments of Pharmacology, (2)Chemistry, (3)Nuclear Medicine, and (4)Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The functional organization of Gbeta is poorly understood. Regions of bovine brain Gbeta that interact with a photoaffinity derivative of an alpha(2)-adrenergic receptor-derived peptide from the third intracellular loop (diazopyruvoyl-modified peptide Q (DAP-Q)) and a hydrophobic membrane probe (3-trifluoromethyl-3-(m-iodophenyl)diazirine (TID)) were examined. We previously showed that DAP-Q cross-links to specific, competable sites on both the alpha and beta subunits of G(o)/G(i) but not on the subunit and that beta subunit was required for stimulation of G(o)/G(i) GTPase activity (Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R.(1994) J. Biol. Chem. 269, 27618-27624). Similarly, we show here that the membrane-associated photoprobe [I]TID labels alpha and beta but not . We have now mapped the sites of incorporation of DAP-Q and TID into the beta subunit. TID labels both the 14-kDa amino-terminal and the 23-kDa carboxyl-terminal fragments from a partial tryptic digest of beta while DAP-Q labels only the carboxyl-terminal fragment. Further mapping with endopeptidase Lys C reveals substantial labeling of multiple fragments by TID while DAP-Q labels predominantly a 6-kDa fragment within the carboxyl-terminal 60 amino acids of beta(1). Thus, regions within the 7th (or possibly 6th) WD-40 repeat of the beta subunit of G protein interact with the receptor-derived peptide while membrane interaction involves multiple sites throughout the beta subunit.


INTRODUCTION

Heterotrimeric G proteins (composed of alpha, beta, and subunits) transmit intracellular signals from a family of plasma membrane-associated G protein-coupled receptors (GPCR). (^1)This family includes adrenergic receptors, photoreceptors, and growth factor receptors among others. Binding of ligand causes a conformational change in the receptor, which activates the associated G protein. The activated G protein dissociates into an alpha subunit and a beta subunit complex. Both the alpha and beta subunits are able to activate intracellular effector enzymes (1) .

The GPCRs have seven transmembrane helices with three cytoplasmic loops and an intracellular carboxyl terminus(2) . Mutagenesis (3, 4) and competition studies using synthetic peptides (5, 6, 7, 8, 9) suggest that the i3 loop and possibly the second cytoplasmic loop and carboxyl-terminal tail are important for receptor-G protein interactions. Peptide Q is a tetradecapeptide from the carboxyl-terminal part of the i3 loop of the alpha(2)AR. Peptide Q can inhibit alpha(2)AR-G(i) coupling and can also mimic GPCRs by binding to and activating G protein directly(5, 8, 10) .

We have previously described a photoaffinity label (DAP-Q) prepared by coupling the sulfhydryl-reactive Br-DAP to the G protein activator peptide (peptide Q)(11) . DAP-Q and a radioiodinated derivative ([I]pHBDAP-Q) cross-link at nanomolar concentrations to specific, competable sites on both the alpha and beta subunits but not on the subunit of G(o)/G(i)(12) . Also, a functional interaction between DAP-Q and beta is required for DAP-Q-stimulated GTPase activity. Binding of DAP-Q to the amino terminus of alpha (12) is consistent with a number of reports implicating the closely associated amino and carboxyl termini of alpha subunits in binding receptor(9, 13, 14, 15, 16, 17) .

In addition to its role in membrane association of alpha subunits(18) , much recent evidence supports a direct interaction of the beta subunit complex in the coupling of receptors to G proteins. Binding of purified beta(1)-adrenergic receptors and rhodopsin with their respective G protein beta subunits has been demonstrated(19, 20) . Kleuss et al.(21, 22) have shown that antisense probes directed against the beta(3) subtype or the (4) subtype can block muscarinic receptor inhibition of calcium currents while probes directed against beta(1) or (3) block somatostatin receptor inhibition of calcium currents(21, 22) . Also, Kisselev and Gautam (23) have shown that rhodopsin binds to a G protein containing (1) but not (2) or (3) subunits. The isoprenoid-modified carboxyl terminus of subunit is important for coupling to rhodopsin(24) .

The beta subunit is composed of seven highly conserved WD-40 repeat regions characterized by a Gly-His followed by 23-41 core amino acids and a WD (Trp-Asp)(25) . The function of these WD-40 repeats is not known, but it has been proposed that the repeats are important for protein-protein interactions(25) . The specific regions of beta subunits participating in either receptor coupling or membrane association remain to be identified. In this report we have mapped the major binding site on the beta subunit for the G protein activator peptide (DAP-Q) and have begun to localize labeling sites for the membrane-associated photoprobe [I]TID.


MATERIALS AND METHODS

Chemicals

Br-DAP and the iodinatable p-hydroxybenzyl derivative of Br-DAP, N-bromoacetyl-N-(4-hydroxybenzyl)-N`-(3-diazopyruvoyl)-1,3-phenylenediamine (pHBDAP), were synthesized as described(11, 12, 26) . pHBDAP was radioiodinated with NaI by the chloramine-T method as described previously(27, 28) . To permit a significant stoichiometry of labeling of beta by [I]pHBDAP-Q to reduce complications of unmodified beta subunits, we isotopically diluted the cross-linker with unlabeled pHBDAP-Q 30-fold from its original specific activity of 2200 to 73 Ci/mmol. [I]TID (10 Ci/mmol) was from Amersham Corp., and the carboxyl-terminal beta subunit antibody (SW/1) was from DuPont NEN. Non-radioactive TID was a gift of Dr. Jonathan B. Cohen (Harvard Medical School). HPLC solvents were from J. T. Baker Inc. All other chemicals and reagents were reagent grade or better and were purchased from Sigma.

Peptide Synthesis

Peptide Q (RWRGRQNREKRFTC) was synthesized by a Biosearch 9600 peptide synthesizer using Fmoc (fluorenylmethoxycarbonyl) chemistry and purified by a preparatory HPLC Beckman System Gold on a reverse phase column. Peptide Q corresponds to the carboxyl-terminal region of the i3 loop of the porcine alphaAR (residues 361-373). The peptide has an additional cysteine attached to the carboxyl terminus of the native receptor sequence. The purity and identity of the peptide was confirmed by mass spectroscopy using a Vestec single quadrupole mass spectrometer with electrospray interface by the Protein and Carbohydrate Structure Core at the University of Michigan.

G Protein Purification and Preparation

G(o)/G(i) was purified from synaptosomal membranes of bovine brain cortex by the method of Sternweis and Robishaw (29) as modified by Kim and Neubig(30) . Protein was quantitated using the method of Schaffner and Weissmann(31) . [S]GTPS binding was measured as described(29) . To obtain purified alpha and beta subunits, the G protein was activated with AlCl(3), MgCl(2), and NaF as described elsewhere(32, 33) . The subunits were then separated as described by Kwon et al.(34) using heptylamine-Sepharose chromatography.

Photolysis of DAP-Q with Purified G Protein Preparations

Peptide Q was conjugated to Br-DAP or [I]pHBDAP, and the complex was purified by HPLC as described previously(11) . DAP-Q or [I]pHBDAP-Q was incubated on ice for 10 min with purified beta subunit or G(o)/G(i) from bovine brain at the indicated concentrations in buffer A (50 mM Na-HEPES, pH 8.0, 1 mM EDTA, 1.3 mM MgCl(2), 0.1% Lubrol, and 60 µM GTP). A 366-nm mineral light (model UVGL-25) was then placed 4.5 cm from the samples for 10 min. SDS-PAGE sample buffer was then added, and cross-linked products were prepared for electrophoresis.

[I]TID Labeling of G Protein in Membranes

G(o) or beta subunits were reconstituted by gel filtration into azolectin vesicles as described(12) . For labeling, 1 µM [I]TID with or without 100 µM unlabeled TID was added to reaction tubes from a stock solution in ethanol. The ethanol was dried with a stream of N(2) in dim light, and then the G protein vesicles were added. Samples were vortexed and then incubated for 10 min on ice prior to photolysis for 30 min as described for DAP-Q (above).

Proteolytic Digestion of the Cross-linked Products

Partial tryptic digestion of beta subunit to cleave at Arg was performed as described(35) . Briefly, purified beta (100 nM or 0.85 µg/lane) was incubated with TPCK-treated trypsin (1:100, w/w) for 5-30 min at room temperature. The reaction was stopped by the addition of soybean trypsin inhibitor (1:10, w/w). Trypsin-treated beta was photolyzed with DAP-Q (1 µM) or [I]TID as described above. The digestion products were analyzed by 16% Tricine gel electrophoresis.

Proteolytic cleavage with Lys C was performed using the Cleveland method(36) . [I]pHBDAP-Q (3 µM) or 1 µM [I]TID was photolyzed with purified beta (14 µM or 50 µg/lane) in 0.1% Lubrol or azolectin vesicles, respectively. The labeled beta subunit was separated from the subunit and non-incorporated label by 12.5% SDS-PAGE. The beta subunit was identified by staining control lanes with Coomassie Blue, and the corresponding regions of the unstained radiolabeled sample were excised. The excised bands were then applied to a 16% Tricine gel in the presence of endoproteinase Lys C (20:1, protein:enzyme), and the gel was electrophoresed according to the method of Cleveland(36) .

SDS-Polyacrylamide Gel Electrophoresis and Western Blots

Cross-linked products were separated on 10 or 12.5% sodium dodecyl sulfate-polyacrylamide gels prepared according to Laemmli (37) or 16% Tricine-polyacrylamide gels(38) . For detection of radioactivity, wet or dried gels were exposed on a Molecular Dynamics PhosphorImager. For Western blots, proteins were transferred to Immobilon, labeled with anti-beta subunit antiserum (SW/1) at 1/1000 dilution, and visualized as described(12) . Alternatively, blots were stained overnight with colloidal gold protein stain (Bio-Rad).


RESULTS AND DISCUSSION

Several lines of evidence indicate that the beta subunit complex binds GPCRs and is important for signal transduction(19, 20, 21, 22, 23, 24, 35) . We recently reported that the photoactive alpha(2)AR-derived i3 loop peptide, DAP-Q, labels a highly specific site within the amino-terminal 17 residues of alpha(o)(12) . DAP-Q also cross-links to a specific site on the beta subunit (12) (see Fig. 1C), and we use this specific labeling to map a potential receptor-interacting region of the beta subunit. In contrast to the specificity of labeling by DAP-Q, labeling of alpha and beta subunits by the membrane-associated photoprobe [I]TID is not blocked by excess unlabeled compound (Fig. 1A). This lack of competition is characteristic of membrane-exposed sites(39, 40) . Interestingly, the subunit did not label with [I]TID (Fig. 1B). While the isoprenoid tail of is required for beta association with membranes(41) , it is possible that the lipid tail is not sufficiently large or reactive to incorporate significant [I]TID, or it may be involved in protein folding or conformation rather than direct lipid interactions.


Figure 1: Incorporation of [I]TID or [I]pHBDAP-Q into G protein subunits. G(o) was labeled in azolectin vesicles with 1 µM [I]TID (A and B) or in Lubrol with 3 µM [I]pHBDAP-Q (73 Ci/mmol) (C) as described under ``Materials and Methods.'' [I]TID-labeled samples (A and B) were separated on a 16% Tricine gel, and [I]pHBDAP-Q-labeled samples (C) were separated on a 10% Laemmli gel. Radioactivity was detected with a PhosphorImager. Excess non-radioactive TID (100 µM) or peptide Q (750 µM) was added where indicated to test specificity. A and B are from the same exposure of a single gel indicating that labeling of subunit is minimal compared with that of alpha and beta.



The functional significance of the specific DAP-Q binding site(s) on beta subunit is supported by the absolute requirement of the beta subunit for stimulation of alpha(o) subunit GTPase activity (12) . Thus, to begin to map the major binding sites on beta for DAP-Q and TID we examined their cross-linking to trypsin-treated beta subunit. Trypsin treatment of native beta subunit results in cleavage of beta at Arg, which generates a 14-kDa amino-terminal fragment and a 23-kDa carboxyl-terminal fragment. Thomas et al.(35) have shown trypsin treatment of native beta subunits does not disrupt tertiary structures or the ability of the complex to associate functionally with the alpha subunit. For this experiment, the ability of DAP-Q to induce a gel shift of the G protein beta subunit was utilized. Fig. 2shows that DAP-Q photolabels the 23-kDa carboxyl-terminal trypsin fragment (A) but not the 14-kDa fragment of the beta subunit (B). It is also evident from Fig. 2A that DAP-Q cross-links equally efficiently to the 23-kDa fragment of trypsin-treated beta and to the uncleaved beta subunit. (^2)In contrast, [I]TID labels both the 14- and 23-kDa fragments (Fig. 2C). Quantitation reveals 23 ± 4% (n = 2) as much labeling of the 14-kDa fragment as the 23-kDa fragment, consistent with significant degrees of membrane contact for both fragments.


Figure 2: Photolabeling trypsin-digested beta subunit with DAP-Q. Purified beta (100 nM or 0.85 µg/lane) in Lubrol (A, B) or azolectin (C) was digested with TPCK-treated trypsin (1:100, w/w) as described under ``Materials and Methods.'' After 30 min, the reaction was stopped by adding soybean trypsin inhibitor (10:1, inhibitor:trypsin). The trypsin digest was then photolyzed with DAP-Q (1 µM) or 1 µM [I]TID. The products were separated by 16% Tricine gel electrophoresis, transferred to Immobilon, and analyzed by Western blot using a carboxyl-terminal anti-beta antibody (A), stained with colloidal gold (B), or scanned with a Molecular Dynamics PhosphorImager (C). The data are representative of four (A and B) or two (C) separate experiments. beta indicates the position of the native (37 kDa) beta subunit, whereas beta and beta(14) indicate the positions of the carboxyl-terminal 23-kDa fragment and the amino-terminal 14-kDa fragment. * indicates the position of a DAP-Q cross-linked product. T.I. indicates the position of soybean trypsin inhibitor.



To further localize the major binding sites on the beta subunit we labeled purified beta with [I]pHBDAP-Q in Lubrol and digested the products with endoproteinase Lys C (20:1, protein:enzyme) according to Cleveland et al.(36) . Fig. 3shows the results of a partial endoproteinase Lys C digestion of [I]pHBDAP-Q-labeled beta subunit. The majority of the radioactivity migrates as a 6-kDa fragment of the beta subunit (lane A, solid arrow). The silver stain of this gel (lane C) reveals a number of beta fragments ranging from 28 to 1 kDa. Complete digestion of the beta subunit should result in fragments ranging from 10.4 to 0.4 kDa. Thus the high molecular weight fragments in lane C show that the beta subunit does not digest to completion under these conditions. Labeling of beta by [I]TID showed a much more extensive distribution of label (Fig. 3, lane B). The 6-kDa fragment incorporated a significant amount of [I]TID, but there were 5 additional fragments clearly labeled.


Figure 3: Partial Lys C digest of purified beta labeled with [I]pHBDAP-Q. Purified beta subunit (14 µM) was photolyzed in the presence of [I]pHBDAP-Q (A and C, 3 µM, 73 Ci/mmol) or [I]TID (B, 1 µM), and the products were electrophoresed on 12% SDS-PAGE. The cross-linked beta subunit was excised, and the gel slices were loaded onto a 16% Tricine gel in the presence of Lys C as described under ``Materials and Methods.'' The polyacrylamide gel was either fixed and exposed to a PhosphorImage screen (A and B) or silver-stained (C). The filled arrow indicates the 6-kDa fragment labeled with DAP-Q, and the open arrows indicate two large fragments, which were not labeled with DAP-Q (see text). The data are representative of three separate experiments.



To identify the position of the 6-kDa fragment within the sequence of the beta subunit, we examined the size of predicted Lys C fragments that overlap the 23-kDa COOH-terminal tryptic fragment of the beta subunit. Only labeling within residues 281-341 in beta(1) or 302-341 in beta(2) predicts labeled fragments smaller than 8.9 kDa (Fig. 4). (^3)Since labeling within 281-301 (light gray in Fig. 4) would generate a radiolabeled fragment of approximately 11.5 kDa from the beta(2) subunit, which is not observed, it is likely that both subunits are labeled within residues 302-341 (black in Fig. 4). However, the 6-kDa apparent molecular mass of the radiolabeled fragment is consistent also with cross-linking to beta(1) in the region of 281-301 provided that cleavage at Lys does not occur.


Figure 4: Linear model of the beta(1) subunit showing cleavage sites and the location of the DAP-Q labeling site. A linear model of beta(1) subunit is shown indicating the 23-kDa carboxyl-terminal trypsin digestion fragment. Within this fragment the Lys C cut sites are shown by residue number following the cut. WD-40 repeat regions are indicated by the numbered boxes. The location of the DAP-Q labeling site is highlighted with the black region being the most likely site while the gray region cannot be absolutely excluded.



The absence of labeling within beta(1)-(128-281) or beta(2)-(128-301) is further supported by the major non-labeled fragments of approximately 16.5 and 19 kDa on the silver stain (Fig. 3C, open arrows). These are similar to the expected masses of 16,671 of beta(1)-(128-280) and 19,077 of beta(2)-(128-301). These partial Lys C digest fragments would be generated if cleavage did not occur at Lys, which may be buried in the fourth WD-40 domain.

In summary, trypsin digest data indicate that the only sites of beta subunit labeling by DAP-Q are within the carboxyl-terminal region of the beta subunit. Lys C digestion shows that the majority of label migrated at 6 kDa. Therefore, the radiolabeled Lys C fragment corresponds to a site within residues 302-341 of either beta(1) or beta(2) or possibly 281-301 of beta(1) (Fig. 4). The region of DAP-Q labeling includes WD-40 repeat 7 and the connecting loop to 6 and possibly repeat 6 itself (Fig. 4). Interestingly, the DAP-Q binding site on beta overlaps with the binding site on beta, which has been shown to include portions in the carboxyl-terminal half of beta(42, 43, 44) .

In contrast to the limited region of contact with the receptor peptide, the membrane contact sites on beta subunit appear to be much more extensive. They encompass both the amino- and carboxyl-terminal tryptic digest fragments, and within these major fragments several small Lys C fragments are labeled. Further mapping will be required to define the details of membrane association as has been done for the Torpedo nicotinic acetylcholine receptor(40) .

Functional data support DAP-Q as an appropriate tool for identifying receptor interaction sites on the beta subunit. Our previous results showed that beta was required for peptide stimulation of alpha(o) GTPase activity(12) , just as is true for receptor activation of purified alpha subunits of G proteins(45) . In addition to the site on beta described in this report, we have shown that DAP-Q labels the amino terminus of alpha(o)(12) . This same NH(2)-terminal region on alpha has been shown to bind to mastoparan (46) and to disrupt rhodopsin-transducin interactions(9) . Although the regions on the beta subunit that bind to alpha are not well defined, Neer and colleagues (47, 48) have shown that residues 204 and 271 in the carboxyl-terminal 23-kDa fragment of beta(1) can be chemically cross-linked to the alpha subunit.

The sequences of beta subunits are highly conserved and display approximately 83% amino acid identity among the known subtypes(49) . In fact, within the region 281-341 only 8 amino acids are different among beta(1), beta(2), and beta(4) (the most abundant subtypes). Are these few differences sufficient for somatostatin and muscarinic receptors to recognize different beta subunits(21) ? As noted above, DAP-Q and the subunit bind to overlapping regions on the beta subunit. Thus, it is possible that the subunit could provide an additional level of specificity to the alpha(2)AR-G protein interaction. In accordance with this hypothesis, somatostatin receptors, muscarinic receptors, and rhodopsin all distinguish between G proteins composed of different subunits (22) . Unlike the beta subunits, subunits display great sequence diversity with only 30% homology between the known subtypes(49) .

This report is the first to define regions of the beta subunit that interact with a receptor-derived G protein activator and a membrane-associated photolabel. Based on these data and that of many other groups, it is likely that the binding site for receptor involves interactions with alpha, beta, and subunits with the carboxyl terminus of beta playing a significant role.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HLGM46417 and GM39561 (to R. R. N.), Michigan Arthritis Center Grant P60-AR20557, and a predoctoral fellowship provided by the American Association of University Women (to J. M. T.). Access to Genetics Computer Group software was provided by National Institutes of Health Grant M01 RR00042. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan, 1301 MSRBIII, Ann Arbor, MI 48109-0632. Tel.: 313-763-3650; Fax: 313-763-4450; :RNeubig{at}umich.edu.

(^1)
The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; Br-DAP, N-bromoacetyl-N`-(3-diazopyruvoyl)-m-phenylenediamine; DAP, diazopyruvoyl; DAP-Q, DAP-modified peptide Q; G(o), abundant G protein purified from bovine brain; G(i), inhibitory G protein purified from bovine brain; pHBDAP, N-bromoacetyl-N-(4-hydroxybenzyl)-N`-(3-diazopyruvoyl)-1,3-phenylenediamine; pHBDAP-Q, pHBDAP-modified peptide Q; i3, third intracellular loop; peptide Q, peptide with the sequence RWRGRQNREKRFTC (amino acids 361-373) from the porcine alpha-adrenergic receptor with additional carboxyl-terminal cysteine; TID, 3-trifluoromethyl-3-(m-iodophenyl)diazirine; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; GTPS, guanosine 5`-3-O-(thio)triphosphate.

(^2)
Notably, both 2- and 4-kDa band shifts are observed for the beta subunit and the 23-kDa fragment. The double incorporation of DAP-Q (4-kDa band shift) is generally observed when cross-linking is done with probe concentrations of 1 µM and above. With lower concentrations only single incorporation (a 2-kDa band shift) is observed(12) .

(^3)
Matrix-assisted laser desorption mass spectral analysis of a Lys C digestion of our purified beta subunit mixture indicated the presence of both beta(1) and beta(2) in our preparation. We observed major fragments of 1730.4 and 2754 daltons, which correspond to N-acetylated beta(1)-(2-15) (expected M(r) of 1729) and N-acetylated beta(2)-(2-23) (expected M(r) of 2753). A minor peak of 2726.8 daltons was also observed, which could either correspond to N-acetylated beta(4)-(2-23) (expected M(r) of 2726) or an incomplete digestion fragment N-acetylated beta(1)-(2-23) (expected M(r) of 2729). The expected masses of labeled fragments were calculated from the mass of the beta subunit fragment plus 965 daltons for the Lys C cleavage product of iodo-pHBDAP-Q (iodo-pHBDAP-Q-(11-14)). Thus, predicted labeled fragments from beta(1) in the observed size range would have masses of 4658 (302-337), 5071 (302-341), 7011 (281-337), and 7424 (281-341), while those from beta(2) would be 4944 (302-337) and 5057 (302-341). The difference in predicted fragment sizes for beta(1) and beta(2) is due to the arginine at position 280 in beta(2) while beta(1) has a cleavable lysine. All other fragments overlapping the carboxyl-terminal 23-kDa tryptic fragment would be larger than 8.9 kDa.


ACKNOWLEDGEMENTS

We thank Dr. Jonathan B. Cohen (Harvard Medical School) for the gift of non-radioactive TID and advice concerning the membrane labeling studies and Dr. Ann Remmers and Partha Mukhopadhyay for assistance in the early work with TID.


REFERENCES

  1. Sternweis, P. C. (1994) Curr. Opin. Cell Biol. 6, 198-203 [Medline] [Order article via Infotrieve]
  2. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang Feng, T. L., Francke, U., Caron, M., Lefkowitz, R. J., and Regan, J. W. (1987) Science 238, 650-656 [Medline] [Order article via Infotrieve]
  3. Strader, C. D., Dixon, R. A. F., Cheung, A. H., Candelore, M. R., Blake, A. D., and Sigal, I. S. (1987) J. Biol. Chem. 262, 16439-16443 [Abstract/Free Full Text]
  4. O'Dowd, B. F., Hnatowich, M., Regan, J. W., Leader, W. M., Caron, M. G., and Lefkowitz, R. J. (1988) J. Biol. Chem. 263, 15985-15992 [Abstract/Free Full Text]
  5. Dalman, H. M., and Neubig, R. R. (1991) J. Biol. Chem. 266, 11025-11029 [Abstract/Free Full Text]
  6. Palm, D., Munch, G., Dees, C., and Hekman, M. (1989) FEBS Lett. 254, 89-93 [CrossRef][Medline] [Order article via Infotrieve]
  7. Konig, B., Arendt, A., McDowel, J. H., Kahlert, M., Hargrave, P. A., and Hofmann, K. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6878-6882 [Abstract]
  8. Ikezu, T., Okamoto, T., Ogata, E., and Nishimoto, I. (1992) FEBS Lett. 311, 29-32 [CrossRef][Medline] [Order article via Infotrieve]
  9. Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B., and Hofmann, K. P. (1988) Science 241, 832-835 [Medline] [Order article via Infotrieve]
  10. Wade, S. M., Dalman, H. M., Yang, S., and Neubig, R. R. (1994) Mol. Pharmacol. 45, 1191-1197 [Abstract]
  11. Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., and Neubig, R. R. (1994) Peptides 15, 829-834 [Medline] [Order article via Infotrieve]
  12. Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R. (1994) J. Biol. Chem. 269, 27618-27624 [Abstract/Free Full Text]
  13. Sullivan, K. A., Miller, R. T., Masters, S. B., Beiderman, B., Heideman, W., and Bourne, H. R. (1987) Nature 330, 758-760 [CrossRef][Medline] [Order article via Infotrieve]
  14. Gutowski, S., Smrcka, A., Nowak, L., Wu, D., Simon, M., and Sternweis, P. (1991) J. Biol. Chem. 266, 20519-20524 [Abstract/Free Full Text]
  15. Palm, I. D., Munch, G., Malek, D., Dees, C., and Hekman, M. (1990) FEBS Lett. 261, 294-298 [CrossRef][Medline] [Order article via Infotrieve]
  16. Shenker, A., Goldsmith, P., Unson, C. G., and Spiegel, A. M. (1991) J. Biol. Chem. 266, 802-808
  17. Simonds, W. F., Goldsmith, P. K., Codina, J., Unson, C. G., and Spiegel, A. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7809-7813 [Abstract]
  18. Sternweis, P. C. (1986) J. Biol. Chem. 261, 631-637 [Abstract/Free Full Text]
  19. Hethier, H., Frohlich, M., Dees, C., Baumann, M., Haring, M., Gierschik, P., Schilta, E., Vaz, W. L. C., Hekman, M., and Helmreich, E. J. (1992) Eur. J. Biochem. 204, 1169-1181 [Abstract]
  20. Phillips, W. J., and Cerione, R. A. (1992) J. Biol. Chem. 267, 17032-17039 [Abstract/Free Full Text]
  21. Kleuss, C., Scherubl, H., Hescheler, J., Shultz, G., and Wittig, B. (1992) Nature 358, 424-426 [CrossRef][Medline] [Order article via Infotrieve]
  22. Kleuss, C., Scherubl, H., Hescheler, J., Schultz, G., and Wittig, B. (1993) Science 259, 832-834 [Medline] [Order article via Infotrieve]
  23. Kisselev, O., and Gautam, N. (1993) J. Biol. Chem. 268, 24519-24522 [Abstract/Free Full Text]
  24. Kisselev, O. G., Ermolaeva, M. V., and Gautam, N. (1994) J. Biol. Chem. 269, 21399-21402 [Abstract/Free Full Text]
  25. Neer, E. J., Schmidt, C. J., Nambudripad, R., and Smith, T. F. (1994) Nature 371, 297-300 [CrossRef][Medline] [Order article via Infotrieve]
  26. Jacob Mosier, G., and Lawton, R. G. (1995) J. Org. Chem. 60, 6953-6958
  27. Roth, J. (1975) Methods Enzymol. 37, 223-233 [Medline] [Order article via Infotrieve]
  28. McConahey, P. J., and Dixon, F. J. (1980) Methods Enzymol. 70, 210-213 [Medline] [Order article via Infotrieve]
  29. Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813 [Abstract/Free Full Text]
  30. Kim, M. H., and Neubig, R. R. (1987) Biochemistry 26, 3664-3672 [Medline] [Order article via Infotrieve]
  31. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514 [Medline] [Order article via Infotrieve]
  32. Roof, D. J., Applebury, M. L., and Sternweis, P. C. (1985) J. Biol. Chem. 260, 16242-16249 [Abstract/Free Full Text]
  33. Bokoch, G. M., Katada, T., Northup, J. K., Ui, M., and Gilman, A. G. (1984) J. Biol. Chem. 259, 3560-3567 [Abstract/Free Full Text]
  34. Kwon, G., Remmers, A. E., Datta, S., and Neubig, R. R. (1993) Biochemistry 32, 2401-2408 [Medline] [Order article via Infotrieve]
  35. Thomas, T. C., Sladek, T., Yi, F., Smith, T., and Neer, E. J. (1993) Biochemistry 32, 8628-8635 [Medline] [Order article via Infotrieve]
  36. Cleveland, D. W. (1983) Methods Enzymol. 96, 222-229 [Medline] [Order article via Infotrieve]
  37. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  38. Schagger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  39. Frielle, T., and Curthoys, N. P. (1983) Biochemistry 22, 5709-5714 [Medline] [Order article via Infotrieve]
  40. Blanton, M. P., and Cohen, J. B. (1994) Biochemistry 33, 2859-2872 [Medline] [Order article via Infotrieve]
  41. Simonds, W. F., Butrynski, J. E., Gautam, N., Unson, C. G., and Spiegel, A. M. (1991) J Biol. Chem 266, 5363-5366 [Abstract/Free Full Text]
  42. Garritsen, A., and Simmonds, W. F. (1994) J. Biol. Chem. 269, 24418-24423 [Abstract/Free Full Text]
  43. Katz, A., and Simon, M. I. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1998-2002 [Abstract]
  44. Yamauchi, J., Kaziro, Y., and Itoh, H. (1995) Biochem. Biophys. Res. Commun. 214, 694-700 [CrossRef][Medline] [Order article via Infotrieve]
  45. Kelleher, D. J., and Johnson, G. L. (1988) Mol. Pharmacol. 34, 452-460 [Abstract]
  46. Higashijima, T., and Ross, E. M. (1991) J. Biol. Chem. 266, 12655-12661 [Abstract/Free Full Text]
  47. Yi, F., Denker, B. M., and Neer, E. J. (1991) J. Biol. Chem. 266, 3900-3906 [Abstract/Free Full Text]
  48. Garcia-Higuera, I., Thomas, T. C., Yi, F., and Neer, E. J. (1995) J. Biol. Chem. 271, 528-535
  49. Spiegel, A. M., Jones, T. L. Z., Simonds, W. F., and Weinstein, L. S. (1994) G proteins , p. 77, R. G. Landes Company, Austin, TX

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