©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
An Intact N Terminus of the Subunit Is Required for the G Stimulation of Rhodopsin Phosphorylation by Human -Adrenergic Receptor Kinase-1 but Not for Kinase Binding (*)

(Received for publication, August 21, 1995; and in revised form, November 6, 1995)

Taraneh N. Haske (1) Antonio DeBlasi (2) Harry LeVine III (1)(§)

From the  (1)Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105 and (2)Centro di Ricerche Farmacologiche e Biomediche, Consorzio Mario Negri Sud, 66030 Chieti, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cleavage after lysine 32 in the G(2) subtype and after lysine 36 in the G(3) subtype of purified mixed brain Gbeta by endoproteinase Lys-C blocks Gbeta-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbeta to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbeta with phospholipids, its ability to support pertussis toxin-catalyzed Galpha(o)/Galpha(i) ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gbeta subunit remains noncovalently associated with the cleaved G fragments. Thus, in addition to recruiting hbetaARK1 to its receptor substrate, G contributes secondary and/or tertiary structural features to activate the kinase.


INTRODUCTION

G-protein-coupled receptor responses to agonist ligands are modulated at multiple levels along the signal transduction pathway. Regulation includes short term effects on receptor coupling and on internalization of the receptor. Longer term effects include down-regulation of the cellular receptor content and mRNA levels (Tholanikunnel et al., 1995). Several different protein kinases have been shown to phosphorylate some of the seven transmembrane helix receptors on cytosolic portions of the molecule, reducing coupling of the receptors to their associated heterotrimeric G-proteins (reviewed by Kobilka(1992)). One family of protein kinases, the G-protein receptor kinases (GRKs), (^1)has been defined, (Premont et al., 1995; Inglese et al., 1993). Three GRKs have been shown to target agonist-occupied receptors, phosphorylating multiple serine/threonine residues adjacent to acidic amino acid residues in the primary amino acid sequence. There are presently six members of this protein kinase family that share a common catalytic domain, diverging in the N- and C-terminal extensions outside of this region.

The GRK2/GRK23 (betaARK1 and betaARK2) subfamilies are encoded by separate genes (Benovic et al., 1991). They were originally shown to phosphorylate the beta(2)-adrenergic receptor and thus were given the name beta-adrenergic receptor kinases or betaARKs. The betaARKs are C-terminally extended relative to other members of the GRK family. The C-terminal 222 amino acids of betaARK1 and betaARK2 contain a domain responsible for the association of the enzyme with heterotrimeric G-protein Gbeta subunits (Pitcher et al., 1992). Addition of Gbeta subunits to an in vitro phosphorylation system stimulates betaARK phosphorylation of receptor substrates (Haga and Haga, 1990, 1992), but not that of peptide substrates (Pitcher et al. 1992). This C-terminal domain of the kinase includes a region homologous to a domain of the platelet protein pleckstrin (PH domain) (Touhara et al., 1993), thought to mediate protein-protein interactions among signaling proteins (Musacchio et al., 1993; Gibson et al., 1994; Ingley and Hemmings, 1994). PH domains may be functionally analogous to the Src homology 2 and 3 domains of tyrosine kinase signaling systems (Mayer et al., 1993). While the portions of the betaARK C terminus involved in the association with Gbeta subunits have been delineated (Koch et al., 1993), less is known about the determinants on the Gbeta partner. The multiple subtypes of Gbeta and G, the requirement for the Gbeta heterodimer for cellular function, (Iniguez-Lluhi et al., 1992), and multiple post-translational modifications (Yamane and Fung, 1993) have impeded study. This paper describes the dissociation of Gbeta binding to hbetaARK1 from Gbeta stimulation of rhodopsin phosphorylation by this kinase after proteolysis with Lys-C. G is cleaved by the protease at lysine 33 in G(2) (lysine 36 in G(3)), and the G fragments remain associated with the Gbeta subunit.


EXPERIMENTAL PROCEDURES

Frozen bovine retinas were from George A. Hormel, Austin, MN. Frozen bovine brain was from Pel-Freez, Rogers, AR. ATP, GDP, GTPS, NAD, sodium cholate, Lubrol PX, isobutylmethylxanthine, L-propanolol, phosphoenolpyruvate, dimyristoyl phosphatidylcholine, forskolin, and pyruvate kinase were from Sigma. Endoproteinase Lys-C from Lysobacter enzymogenes (catalog no. 476986) was from Boehringer Mannheim. Reagents for SDS-PAGE were from Research Organics. Nitrocellulose (BA83) was obtained from Schleicher and Schuell. DE-52 was from Whatman. Antibodies specific for beta(1), beta(2), beta(3), beta(4), pan-beta, (2), (3), (5), and (7) of the heterotrimeric G-protein Gbeta and neutralizing peptides were purchased from Santa Cruz Biotechnology. These antibodies are also useful for enzyme-linked immunosorbent assay determinations. Antibodies to the C-terminal 222 amino acids of human betaARK1 were raised against the glutathione S-transferase (GST) fusion protein (Cocalico Biologicals, Reamstown, PA) and purified from an IgG fraction following adsorption against immobilized GST by affinity chromatography on immobilized GST-C-terminal 222-amino acid human betaARK1, eluting with 0.1 M glycine, pH 2. Secondary antibody (donkey anti-rabbit peroxidase) and ECL detection reagents were purchased from Amersham. [-P]ATP (3000 Ci/mmol), [-S]GTP (1045 Ci/mmol), and [alpha-P]NAD (30 Ci/mmol) were from DuPont NEN. S-Adenosyl-L-[^3H-methyl]methionine (77 Ci/mmol), the I-cAMP Scintillation Proximity Assay Kit, Rainbow prestained molecular weight markers, and Amplify were acquired from Amersham. Purified recombinant human betaARK His(6) (PH+C) (Gly-Ser) protein was generously provided by Dr. Daruka Mahadevan (Mahadevan et al., 1995). DNA encoding the betaARK C-terminal domains betaARK (PH+C) (Gly-Ser) and the C-terminal 222 amino acids (Pro-Leu) were cloned by PCR from the human betaARK1 cDNA provided by Dr. A. DeBlasi (Chuang et al., 1992), and their nucleotide sequences confirmed on an Applied Biosystems model 373A automated sequencer. The glutathione S-transferase fusion proteins were produced in Escherichia coli strain BL21(DE3) using the pGEX-2T vector system (Pharmacia Biotech Inc.) and purified according to standard protocols. Peptides corresponding to residues 8-34 of G(2) or residues 3-29 of Gbeta(2) were kindly provided by Dr. R. Neubig, Department of Pharmacology, University of Michigan.

Heterotrimeric G-proteins were isolated from frozen bovine brain, and the Gbeta subunit complex (mixed subtypes of beta and subunits) purified by chromatography in sodium cholate on heptylamine-Sepharose (Sternweis and Pang, 1990) in the presence of GDP/AlMgF. Further resolution of the separated Gbeta from Galpha subunits for ADP-ribosylation studies was achieved by ion exchange chromatography on a MonoQ (Pharmacia) column in 0.1% (w/v) Lubrol PX (Sternweis and Pang, 1990). The purified Galpha and Gbeta subunits were stored in aliquots at -80 °C. Two different batches of Gbeta when assayed for effects of Lys-C proteolysis on rhodopsin phosphorylation and hbetaARK1 binding activity revealed no discernible differences. Carboxymethylation of the C terminus of the subunit of Gbeta with S-adenosyl-L-[^3H-methyl]methionine was accomplished using a cholate-extracted brain membrane fraction (Fung et al., 1990). Bovine retinal rod outer segments (ROS) containing rhodopsin were purified under red light illumination and urea-extracted before use as a phosphorylation substrate (Phillips et al., 1989). SDS-PAGE was performed in 10% acrylamide, 0.267% bisacrylamide gels containing 0.1% SDS in the Laemmli buffer system (Laemmli, 1970). Rhodopsin phosphorylation was determined after separation of P-labeled proteins by SDS-PAGE. Radioactive gels were fixed for 10 min in 25% methanol, 10% acetic acid, washed with distilled water for 10 min, and the gels dried at 80 °C under vacuum before exposure to Kodak XAR-5 or X-Omat-LS film. The P radioactivity of the rhodopsin band was quantitated from the film using a BioImage (Millipore) scanner. Tritiated proteins separated by SDS-PAGE were visualized after fixation in 25% methanol, 10% acetic acid, soaking in an Amplify enhancement solution for 45 min, and drying and exposing to film at -80 °C for 3-7 days. betaARK1 and Gbeta were determined after transfer of polypeptides separated by SDS-PAGE on a 10% acrylamide, 0.367% bisacrylamide in the Laemmli buffer system (Laemmli, 1970) to 0.2-µm pore size nitrocellulose (LeVine and Sahyoun, 1988). An affinity-purified antibody directed against the C-terminal 222 amino acids of hbetaARK1 recognizing the PH domain of that kinase (Sallese et al., 1995) was used to detect the enzyme. Gbeta subunits were detected with pan-beta subunit-specific antibodies. beta and subunits of the heterotrimeric G-proteins were separated on 16.5% acrylamide, 0.5% bisacrylamide gels containing 6 M urea and 0.1% SDS in a Tris-Tricine buffer system (Schagger and von Jagow, 1987). The subunits were transferred as described for betaARK1 and were detected with Gbeta- or G- subtype subunit-specific antibodies. Quantitation of the ECL was standardized as described (Mahadevan et al., 1995).

Measurement of the Ability of Gbeta to Stimulate Rhodopsin or Synthetic Peptide Phosphorylation by hbetaARK1

Gbeta subunits (20-1400 nM) were incubated in a 60-µl reaction volume containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 6 mM MgCl(2), 16 µCi/ml [-P]ATP, 50 µM ATP, 10 µM GTPS, and 160 pmol of rhodopsin as urea-extracted ROS. After 10 min on ice, 5 µl of 50 µg/ml recombinant hbetaARK1 purified from Sf9 cells were added, and the reaction was illuminated with a fluorescent table lamp at 30 °C for 20 min. The rate of phosphorylation was linear for at least 30 min under these conditions. The reaction was terminated with SDS-sample buffer without boiling, and the proteins resolved by SDS-PAGE. Phosphorylation of the synthetic peptide RRREEEEESAAA (Immunodynamics, Inc., La Jolla, CA) was carried out in the same reaction buffer with 100 µM peptide in place of ROS. The reaction was stopped by the addition of 0.5 volume of 30% trichloroacetic acid. Phosphorylated products were spotted onto Whatman P-81 phosphocellulose paper and washed with 75 mM phosphoric acid three times for 15 min each to remove the free nucleotide. Phosphorylated peptide was determined by liquid scintillation counting of the paper (Cook et al., 1982). Translocation of hbetaARK1 was performed according to Chuang et al.(1992) and involved a 3-min incubation of 300 pmol of rhodopsin (as urea-treated ROS), 5 µl of 50 µg/ml hbetaARK1, and absence or presence of 178 nM Gbeta under a fluorescent table lamp at room temperature in 20 mM Tris-HCl, pH 7.8, 2 mM EDTA, 10 mM MgCl(2), 2 mM dithiothreitol. The ROS were pelleted at 4 °C at 109,000 times g for 10 min in a TLA 100.3 rotor with microcentrifuge tube adapters. The pellets were resuspended in 50 µl of 20 mM Tris-HCl, pH 8.0, and assayed as above for rhodopsin phosphorylation.

Gbeta Subunit-mediated ADP-ribosylation of Galpha(o) and Galpha(i) by Pertussis Toxin

The ability of Gbeta subunits to support pertussis toxin-catalyzed [P]ADP-ribose transfer from [alpha-P]NAD to Galpha(o)/Galpha(i) was determined essentially as described by (Kwon et al., 1993), with 5 µM NAD (2 µCi/assay), 0.5 mM dimyristoyl phosphatidylcholine, and 7.5 µg/ml activated pertussis toxin in a final volume of 50 µl of 75 mM Hepes buffer, pH 8.0, 2 mM dithiothreitol, 1 mM MgCl(2), 1 mM EDTA, and 10 µM GDP. The dose response for Gbeta subunits was determined at a constant Galpha(o)/Galpha(i) concentration. After SDS-PAGE, the incorporation of P into polypeptides migrating in the Galpha region was determined by densitometry from autoradiograms.

Adenylate Cyclase Inhibition by Gbeta Subunits

Dose-dependent inhibition of platelet membrane type I adenylate cyclase by Gbeta subunits was determined as described by Kwon et al.(1993), except that cAMP was determined by Scintillation Proximity immunoassay (Amersham) according to the manufacturer. Dilutions of Gbeta subunits were made in 20 mM Tris-HCl, pH 7.6, 0.1% (v/v) Lubrol PX. Reactions were run in a 100-µl final volume containing 25 mM Tris-HCl, pH 7.6, 2.5 mM EGTA, 2 mM MgCl(2), 0.1 mM isobutylmethylxanthine, 10 µM propanolol, 0.2 mM ATP, 0.01 mM GTP, 0.8 mM phosphoenolpyruvate, 10 µM forskolin, and 10 µg/ml pyruvate kinase. Ten microliters of diluted Gbeta subunits were preincubated on ice in the reaction mixture with 5 µl (15 µg) of platelet membrane protein for 5 min. The tubes were transferred to a 30 °C water bath and the incubation continued for 15 min. One hundred microliters of ice-cold 5% (w/v) trichloroacetic acid were added to stop the reaction on ice. Cyclic AMP content was determined on aliquots of the supernatant after centrifugation at 16,000 times g for 10 min at room temperature to remove precipitated material. Adenylate cyclase activity was linear in both membrane protein and time over the ranges used.

Proteolysis of Gbeta by Endoproteinase Lys-C

Cleavage of the Gbeta complex by Lys-C protease was performed in 50 mM Tris-HCl, pH 8.6, 1 mM dithiothreitol, 0.1 mM EDTA, 0.8% cholate, 0.05% Lubrol PX for 30 min at 30 °C using a protease:protein ratio of 1:10 (w/w). The digestion was terminated by the addition of 20 µg/ml leupeptin. To facilitate comparison of protease treatment, the different functional assays were carried out with the same batches of Gbeta stored at -70 °C that had been kept on ice, incubated without protease at 30 °C, incubated with leupeptin-inactivated protease at 30 °C, or incubated with active Lys-C at 30 °C for 30 min. Thirty minutes of incubation of Gbeta at 30 °C without protease had no discernible effect on its measured biochemical properties.

Binding of Gbeta Subunits to PH Domain-containing C-terminal betaARK1 Fragments

Gbeta subunit binding to immobilized PH domains was determined by immunoblot analysis with pan-anti-beta subunit antibodies as described by Mahadevan et al.(1995).

Amino Acid Sequencing of Lys-C-cleaved Gbeta

Separation of the Gbeta and G subunits was achieved by C4 reverse phase chromatography under the conditions used to separate the farnesylated subunit of transducin on a C18 column (Parish and Rando, 1994). Western blot analysis showed that the Gbeta and G subunits were separated under these conditions (data not shown). Fifty µg of Lys-C-treated Gbeta (1.2 nmol) was diluted with an equal volume of 6 M guanidine HCl, 50 mM dithiothreitol and injected onto a Brownlee 4.6 times 30-mm 300-Å pore size C4 reverse phase column equilibrated with 5% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid at 0.75 ml/min. After a 10-min wash period, the proportion of acetonitrile was linearly increased to 65% over 40 min and held at 65% for 5 min. The effluent was monitored during the separation at 214 nm, and 0.75-ml fractions were collected. Fractions containing the subunits as judged by comigration of digested [^3H]carboxymethylated Gbeta (on a separate HPLC run) and the appearance of new 214 nm absorbing peaks were reduced to near dryness under vacuum on a Savant SpeedVac. The fractions were subjected to 15 cycles of automated Edman degradation on an Applied Biosystems 477A protein sequencer and the phenylthiohydantoinamino acids identified with the integral HPLC unit of the sequencer.


RESULTS

The Gbeta subunits of the heterotrimeric G-proteins form a heterodimer (beta = 35 kDa or 36 kDa, = 6748-8321 kDa), depending on the subunit subtypes and post-translational modifications. This non-covalent complex is not dissociable under non-denaturing conditions and associates reversibly with an undetermined variety of Galpha subtypes and with a protein kinase A substrate, phosducin (Bauer et al., 1992), as well as a host of effector molecules such as adenylate cyclase, ion channels, and phospholipases (Clapham and Neer, 1993). The Gs are multiply post-translationally modified by N termini N-acetylation (Wilcox et al., 1994) and C-terminal cysteine geranylgeranylation and carboxymethylation (reviewed by Yamane and Fung(1993)). Although these modifications are not required for assembly of the Gbeta dimer, prenylation apparently modulates interactions with other proteins (Kisselev et al., 1994) and their interaction with the lipid bilayer (Muntz et al., 1992; Iniguez-Lluhi et al., 1992). Gbeta requires small amounts of detergent to remain in solution.

Lys-C Proteolysis of Gbeta

The structure of the Gbeta dimer is compact, judging from its hydrodynamic properties (Huff and Neer, 1986) and by the resistance of the native protein to proteases such as trypsin (Fung and Nash, 1983; Tamir et al., 1991; Winslow et al., 1986). Proteolysis by the endoproteinase Lys-C from L. enzymogenes was carried out in sodium cholate, a detergent with a low aggregation number (small number of detergent molecules per micelle) (Calbiochem, 1993) to minimize interference with the hydrophobic C terminus of Gbeta.

Reducing SDS-PAGE analysis of the proteolytic product as a function of protease:Gbeta ratio revealed a size range for the G fragment of M(r) 3400-5200 by silver staining, a reduction from M(r) 6900-8600, corresponding to an M(r) of 3500. There was no discernible effect on the M(r) of the Gbeta subunit (Fig. 1). The mixed Gbeta preparation isolated from frozen bovine brain contains multiple subtypes of Gbeta and G subunits, the latter accounting for the smear around M(r) 6500. Antibodies raised to synthetic peptides corresponding to the N-terminal 17 residues of the G subunits or 21 amino acids of the Gbeta subunits (Santa Cruz Biotechnology) are subtype-selective and can be used to determine the distribution of the subtypes in the preparation. Western immunoblotting of two separate purifications revealed that the purified bovine brain Gbeta used in these studies has the following distribution of Gbeta and G subunits: beta(4) beta(2) > beta(1) > beta(3); (2) (3) (5) > (7) (data not shown). This quantitation assumes that the subtype-selective antibodies are equally capable of detecting antigen at 1.6 µg/ml antibody. In addition, the purification of the Gbeta subunits could influence the recovery and thus the apparent distribution of the different subtypes.


Figure 1: 16% acrylamide, 6 M urea Tris-Tricine SDS-PAGE analysis of the cleavage of Gbeta by Lys-C (silver-stained). 2 µg of Gbeta were digested with the indicated ratio of Lys-C protease:Gbeta under the conditions described under ``Experimental Procedures.'' Lane 1, no protease; lane 2, 1:400; lane 3, 1:200; lane 4, 1:100; lane 5, 1:50; lane 6, 1:25; lane 7, 1:12.5. The Gbeta and G subunit positions are indicated by brackets.



Methylation of the C-terminal cysteine (Cys) carboxyl moiety of the purified brain Gbeta with S-adenosyl-L-[^3H-methyl]methionine (Fung et al., 1990) provided a marker for the C terminus of the G subunit. Lys-C treatment of the tritiated Gbeta released a labeled fragment of M(r) 3500 (Fig. 2), mirroring the shift on SDS-PAGE to lower M(r) seen with silver staining (Fig. 1). This implies that the extreme C terminus of the G subunit remains intact. Immunoblotting after SDS-PAGE showed that N-terminal G subunit immunoreactivity (residues 2-17) was eliminated by Lys-C treatment (Fig. 3). By contrast, for the Gbeta subunit, neither the N-terminal immunoreactivity (data not shown) nor the size on SDS-PAGE (Fig. 1) was affected by Lys-C treatment. Gel permeation chromatography of the [^3H]carboxymethylated Gbeta on a Superose 12 column in 0.8% cholate before or after Lys-C treatment demonstrated that the fragments of the cleaved G subunit remained complexed with the Gbeta subunit (Table 1). When the Superose 12 column fractions were assayed by enzyme-linked immunosorbent assay and slot blotting onto nitrocellulose, virtually no loss of the immunoreactive N-terminal fragment(s) of Gbeta was observed. By contrast, denaturing SDS-PAGE and subsequent Western blot analysis of the same fractions revealed a significant loss of N-terminal G subunit immunoreactivity (Table 1).


Figure 2: Reduction in M(r) of C-terminal [^3H]carboxymethylated bovine brain Gbeta by Lys-C proteolysis visualized by ^3H autoradiography. 100 µg of bovine brain Gbeta were carboxymethylated with 0.5 mg of cholate-extracted bovine brain membrane protein and 50 µCi of S-adenosyl-L-[^3H-methyl]methionine (77 Ci/mmol) in 0.5 ml of carboxymethylation buffer and the labeled proteins re-extracted with cholate as described under ``Experimental Procedures.'' Lane 1, [^3H]carboxymethylated proteins incubated for 30 min at 30 °C in the absence of protease; lane 2, + 2 µg of endoprotease Lys-C. The M(r) 24,000 labeled protein is an endogenous substrate for the carboxymethyltransferase derived from the brain membrane enzyme source. This contaminant represents a minor fraction of the ^3H label incorporated. Lys-C treatment leads to a M(r) 3500 decrease in size of the labeled G subunit. The leading ion front ran to the very bottom of the gel.




Figure 3: Removal of N-terminal G subunit immunoreactivity by Lys-C proteolysis. A total of 10 µg of bovine brain Gbeta was incubated with the indicated ratio of endoprotease Lys-C for 30 min at 30 °C. The control lane was material incubated without Lys-C. The digest was then separated by Tris-Tricine-urea SDS-PAGE and transferred to nitrocellulose as described under ``Experimental Procedures.'' The blot was reacted with a 1:1 mixture of G-antibodies specific for the N-terminal residues(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) of the G(2) and G(3) subtypes and the immunoreactivity quantitated as referenced under ``Experimental Procedures.'' The experiment was repeated twice as single determinations with similar results. A, raw film data. Lane 1, 1:4 (Lys-C:Gbeta (w/w)); lane 2, 1:8; lane 3, 1:16; lane 4, 1:32; lane 5, no Lys-C added. B, densitometric scan of film.





Amino Acid Sequence of the Lys-C-treated G Subunit

Gbeta subunits were digested with Lys-C, dissociated with 3 M guanidine hydrochloride + 50 mM dithiothreitol, and separated by C4 reverse phase chromatography as described under ``Experimental Procedures.'' Fig. 4shows the migration of the [^3H]carboxymethyl marker of the C terminus of the G subunit on a separate HPLC run. The arrow marks the position of the truncated C-terminal G sequences (Ala-Cys for G(2); Ala-Cys for G(3)). The two G subtypes nearly comigrated on reverse phase chromatography so their sequences were determined simultaneously. This was possible because both sequences are known, they were present in different amounts, and they differ at several positions. The asterisk shows the position of the undigested G(2) and G(3) polypeptides, while the cluster of radioactive peaks around fraction 40 represent undissociated Gbeta and the unidentified carboxymethylated M(r) 24,000 protein in brain membranes (see Fig. 1and Fung et al.(1990)). Table 2shows the sequences obtained from the major peaks of 214 nm absorbance produced by Lys-C treatment of unlabeled isolated Gbeta subunits. G(2), the major immunoreactive G subtype in the purified brain Gbeta preparation, was likewise the prominent proteolytic fragment represented. Both G(2) and G(3) yielded peptides beginning with Ala (numbering system of G(2), A36 of G(3)) immediately following Lys, consistent with the cleavage specificity of Lys-C after lysine. The [^3H]carboxymethylation experiments indicate that this C-terminal peptide extends to the modified penultimate cysteine. The removal of 32(35) amino acid residues is consistent with the M(r) change in G seen by SDS-PAGE (3500, Fig. 1and Fig. 3). G sequences corresponding to other potential Lys-C proteolytic fragments of G(2), G(3), or other known G subtypes were not detected. Minor 214 nm absorbance peaks contained short C-terminal G fragments that were unrelated to Lys-C cleavage specificity and probably represent co-purified proteolytic fragments in the initial brain preparation. Small amounts of sequences corresponding to N-terminal methionine-containing and non-acetylated G(2) and G(3) were also detected. The same sequencing results were obtained for two independent preparations of Gbeta subunits.


Figure 4: C4 reverse phase chromatography of [^3H]carboxymethylated Gbeta subunits. Fifteen µg of [^3H]carboxymethylated Gbeta were treated with either buffer or 1 µg of Lys-C in a final volume of 100 µl for 1.5 h at 30 °C. The samples were dissociated with guanidine hydrochloride and subjected to reverse phase chromatography as described under ``Experimental Procedures.'' The ^3H content of the 0.75-ml fractions collected was determined by liquid scintillation counting. Circles, incubated without protease; squares, + 1 µg of Lys-C. Arrow, C-terminal G peptides (see text); asterisk, undigested G(2), G (3) polypeptides.





Functional Effects of Cleavage of the G Subunit: Biochemical Activities of Gbeta Subunits

Since Gbeta subunits do not possess an intrinsic enzymatic activity, the effect of Lys-C truncation on several extrinsic measures of functionality of the treated and control Gbeta subunits was evaluated. Their ability to bind to ROS and to modulate rhodopsin phosphorylation by full-length recombinant hbetaARK1 was measured. Their ability to bind to the GST-betaARK C-terminal domain of 222 amino acids (Pro-Leu) and to bind to a more restricted region of the kinase, GST- or His(6)-tagged betaARK PH+C domain (betaARK G556-S670), was determined. Finally, the ability of the treated and control Gbeta subunits to associate with and to modulate Galpha subunit activities and to regulate the activity of an effector, Type I adenylate cyclase, were also assessed.

Modulation of betaARK1-mediated Receptor Phosphorylation

The Lys-C-treated Gbeta subunit preparation supported substantial translocation of human betaARK1 to isolated retinal rod outer segments (Fig. 5A), but failed to stimulate phosphorylation of rhodopsin (Fig. 5B). Gbeta subunits treated with leupeptin-inhibited Lys-C fully supported both translocation and phosphorylation. Fig. 5C shows that Lys-C proteolysis of G(2) and G(3) abrogates stimulation of betaARK1 phosphorylation of receptor substrates without abolishing binding of the kinase. The saturation of the dose response of phosphorylation observed at low concentrations of Lys-C-digested Gbeta contrasts with the roughly linear increase with control incubated Gbeta. The low stimulation seen in the Lys-C-treated Gbeta may be residual undigested Gbeta, or it may represent an intrinsically lower stimulatory activity. Phosphorylation of rhodopsin was linearly related to the amount of betaARK1 and Gbeta present in the assay. The lack of effect of Gbeta subunits on synthetic peptide substrate RRREEEEESAAA was unaltered by proteolysis (data not shown).


Figure 5: Modulation of betaARK1-mediated receptor phosphorylation. Incubations of Gbeta subunits with ROS and betaARK1 and phosphorylation were performed as described under ``Experimental Procedures.'' A, translocation of betaARK1 to rod outer segment membranes containing rhodopsin. The amount of betaARK1 binding to light-activated urea-extracted ROS in the absence, presence of Gbeta subunits, and presence of Lys-C digested Gbeta subunits was determined after SDS-PAGE by immunoreactivity with 0.1 µg/ml anti-betaARK1 antibody. Quantitation of the ECL reaction was by densitometry. Raw film data are shown at the top. Lane 1, translocation of betaARK1 to ROS in the absence of Gbeta subunits; lanes 2 and 3, Lys-C digested Gbeta subunits; lanes 4 and 5, Gbeta subunits treated with Lys-C in the presence of 10 µg/ml protease inhibitor leupeptin. The experiment was repeated three times with similar results. B, lack of stimulation of rhodopsin phosphorylation by proteolyzed Gbeta subunits. betaARK1 phosphorylation of rhodopsin in ROS was determined after translocation of the kinase in the absence, presence, and presence of Lys-C-digested Gbeta subunits. Following SDS-PAGE, the P incorporated into rhodopsin was quantitated by densitometry of the autoradiogram. C, dose response of Gbeta subunits on rhodopsin phosphorylation. ROS were phosphorylated with betaARK1 and the indicated concentrations of control incubated or Lys-C digested Gbeta subunits as indicated under ``Experimental Procedures'' without removing unbound proteins. Following SDS-PAGE the P incorporated into rhodopsin was quantitated by densitometry of the autoradiogram. Circle, control incubated Gbeta subunits; square, Lys-C digested Gbeta subunits. Data are single determinations, and the experiment was repeated twice with similar results. The raw autoradiographic data are shown at the top. The first five lanes are Gbeta subunits treated with Lys-C in the presence of 10 µg/ml leupeptin, while the final four lanes are Lys-C-digested Gbeta subunits. Lane 1, no Gbeta subunits; lanes 2 and 6, 27.9 nM Gbeta subunits; lanes 3 and 7, 55.8 nM; lanes 4 and 8, 112 nM; lanes 5 and 9, 223 nM.



Binding of Gbeta Subunits to the betaARK1 PH Domain

There was no notable difference between Lys-C-treated and untreated Gbeta subunit association with the PH domain contained within the C-terminal 222-amino acid fragment of hbetaARK1 (GST-(P466-L689)) (Fig. 6), or the shorter (PH + C-terminal helix) (G556-S670) GST-fusion or His(6)-tagged (PH+C) domains (data not shown) over a range of Gbeta concentrations. Thus, Lys-C proteolysis of G does not detectably alter association of Gbeta subunits with the kinase in the absence of the catalytic and N-terminal sequences of hbetaARK1.


Figure 6: Effect of Lys-C proteolysis of Gbeta subunits on binding to the isolated PH domain of betaARK1. The indicated concentrations of treated Gbeta subunits were incubated in a 50-µl reaction volume with 0.5 µM GST-betaARK1 C-terminal (Pro-Leu) fusion protein immobilized on glutathione-Sepharose as indicated under ``Experimental Procedures.'' The beads were washed and subjected to SDS-PAGE, and the proteins transferred to nitrocellulose for immunodetection of the Gbeta with a pan-Gbeta-specific antibody. A, raw film data. Lanes 1, 5, and 9, 106 nM Gbeta subunits; lanes 2, 6, and 10, 319 nM; lanes 3, 7, and 11, 957 nM; lanes 4, 8, and 12, 2874 nM. Lanes 1-4 represent Gbeta subunits incubated on ice, lanes 5-8 are Gbeta subunits treated with Lys-C at 30 °C for 30 min in the presence of 10 µg/ml leupeptin and lanes 9-12 are Gbeta subunits treated at 30 °C with Lys-C for 30 min. B, quantitation by densitometry. Circle, Gbeta subunits incubated on ice; square, Gbeta subunits treated with Lys-C at 30 °C for 30 min in the presence of 10 µg/ml leupeptin to inhibit the protease; triangle, Gbeta subunits treated at 30 °C with Lys-C for 30 min. Data are representative of four experiments with single determinations.



Effects of N-terminal Gbeta and G Synthetic Peptides on Gbeta Subunit Interactions with hbetaARK1

Antibodies (Santa Cruz Biotechnology) to N-terminal residues(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) of G ((2) or (3)) or the N-terminal region of Gbeta (residues 25-40 of beta(2) or beta(4); 20 µg/ml antibody) or the synthetic peptides in 1 mg/ml gelatin supplied by Santa Cruz Biotechnology) corresponding to these regions (40 µg/ml, 25 µM) were used to try to block or enhance betaARK1 binding to Gbeta to immobilized PH domains or to block or stimulate betaARK1 phosphorylation of rhodopsin in ROS without significant effect (data not shown). Similar results were seen for peptides corresponding to residues 8-34 of G(2) or residues 3-29 of Gbeta(2) (200 µM each). This lack of effect suggests either that the most N-terminal segments of G and Gbeta are not involved in the assayed interactions with betaARK1 or that the interactions with the free peptides are of low affinity.

Catalysis of Pertussis Toxin-mediated ADP-ribosylation of Galpha Subunits

Pertussis toxin, an ADP-ribosylating enzyme, modifies a cysteine in the Galpha(o) and Galpha(i) family of G-proteins only when the Galphabeta heterotrimer is formed (Neer et al., 1984). Isolated mixed Galpha(i)/Galpha(o) from brain was titrated with proteolyzed and control Gbeta subunits. The reconstituted G-proteins were treated with pertussis toxin as described under ``Experimental Procedures.'' A dose-dependent increase to saturation was observed with increasing amounts of Gbeta subunits added to Galpha subunits. For the particular amount of Galpha(i)/Galpha(o) used, saturation was achieved around 600 nM Gbeta subunits. No significant difference was noted for any of the treatments of the Gbeta subunits (Fig. 7A). Heating of the Gbeta subunits at 65 °C for 10 min abolished their ability to support ADP-ribosylation of Galpha subunits.


Figure 7: Functional assay of truncated Gbeta subunits. A, Gbeta subunit-mediated ADP-ribosylation of Galpha(o)/Galpha(i) by pertussis toxin. The reaction was carried out as described under ``Experimental Procedures.'' Results are expressed as average of duplicates ± S.D. The experiment was repeated twice with similar results. Circle, Gbeta subunits incubated on ice; square, Gbeta subunits treated with Lys-C at 30 °C for 30 min in the presence of 10 µg/ml leupeptin to inhibit the protease; triangle, Gbeta subunits treated at 30 °C with Lys-C for 30 min. B, inhibition of forskolin-stimulated platelet type I adenylate cyclase by Gbeta subunits. The effect of treated Gbeta subunits on platelet membrane adenylate cyclase activity was determined as described under ``Experimental Procedures.'' Circle, Gbeta subunits incubated on ice; square, Gbeta subunits treated with Lys-C at 30 °C for 30 min in the presence of 10 µg/ml leupeptin to inhibit the protease; triangle, Gbeta subunits treated at 65 °C for 10 min to inactivate the proteins. Data shown are the average of triplicates ± S.D. Basal adenylate cyclase activity = 200 pmol/min/mg protein, forskolin-stimulated adenylate cyclase activity (100%) = 2200 pmol/min/mg protein. The experiment was repeated twice.



Inhibition of Platelet Adenylate Cyclase by Gbeta Subunits

The type I adenylate cyclase is inhibited by Gbeta subunits at a site distinct from that modulated by Galpha (Taussig et al., 1994). Titration of forskolin-stimulated platelet membrane Type I adenylate cyclase activity with Gbeta stored on ice, Gbeta subunits treated with Lys-C inhibited with leupeptin, and Lys-C-treated Gbeta subunits were indistinguishable (Fig. 7B). Proteolytic nicking of the G subunit, therefore, did not detectably alter the adenylate cyclase modulatory function of Gbeta subunits.


DISCUSSION

Gbeta subunits participate in a diverse range of biological interactions, from anchoring and modulating the GTPase activity of Galpha subunits, to activation or inhibition of transmembrane signaling effectors such as phospholipase C beta and isozymes, phospholipase A(2), adenylate cyclase isozymes, and ion channels (reviewed by Clapham and Neer(1993)). They are also critically involved in regulating the activity of several of the G-protein coupled receptor kinases (GRK2 and GRK3) toward receptor substrates (Inglese et al., 1993). The multiple isotypes (5-beta and 7- isotypes) of Gbeta subunits provide a daunting array of possible combinations, which may be significant (Kleuss et al., 1993; Kleuss et al., 1992). The assembly of the dimeric structure is further complicated by their subsequent extensive post-translational modification and proteolytic processing. Chimeric studies in COS7 cells taking advantage of the differential interaction of G(1) with Gbeta(1) but not Gbeta(2), while G(2) will interact with both Gbetas (Simonds et al., 1991), defined multiple sites of interaction of the beta subunit with the subunit, and assigned subtype selectivity to Gbeta(1) residues 215-340 (Garritsen and Simonds, 1994) or residues 210-293 (Katz and Simon, 1995). A series of G(2) truncation experiments suggest that a region between residues 45-59 in the G subunit are involved with dimerization with Gbeta(1) (Mende et al., 1995).

At high concentrations trypsin will cleave the Gbeta subunit at Arg, generating two proteolytic fragments of M(r) 26,000 and 15,000 without noticeable effect on the G subunit in native Gbeta subunits (Tamir et al., 1991). The accessibility of the cleavage site in the subunit to Lys-C proteolysis demonstrated in the present work illuminates the quaternary structure of the Gbeta dimer. To minimize possible interactions of the prenyl moiety with lipid bilayers and potential steric hindrance, the proteolysis was performed in cholate, an anionic detergent with the small aggregation number of 2 cholate molecules/micelle (Calbiochem, 1993). There are 7 (G(2)) or 6 (G(3)) lysines in the G subunit, which could potentially be available for Lys-C proteolysis. The major identified G products of this protease begin with Ala (G(2)) and Ala (G(3)). Monitoring of the migration of the C-terminal [^3H]carboxymethylated G on HPLC gave no evidence for specific smaller or larger cleavage products. Thus, the majority of the treated G retains the prenylated C-terminal cysteine residue. This part of the molecule is thought to interact with the seven-transmembrane helix receptor in the fashion of transducin of the visual system. The farnesylated C terminus of G(t)beta(1)(1), stabilizes the active MII form of rhodopsin, which activates G(t) (transducin) in the presence of GTP (Kisselev et al., 1994). Hints as to the relative positioning of the beta and subunits come from several sources. Copper o-phenanthroline-mediated cross-linking of Gbeta(1) and G(t)(1) through proximal intersubunit cysteine residues in transducin indicates that beta Cys and Cys or Cys are nearby in the three-dimensional protein structure (Bubus and Khorana, 1990). This was interpreted as a point of close contact between the subunits. Alignment of transducin G(1) with G(2) and G(3) places the reactive Cys/Cys at the site of Lys-C digestion determined here. Perhaps lysines C-terminal to this position are not accessible to the protease because of beta subunit- subunit interactions or interactions involving the prenyl group. A series of sequential amino acid replacements in G(2)(35-37) and G(1)(38-40) are sufficient to specify the appropriate beta- selectivity (Lee et al., 1995). Interestingly, this motif is immediately C-terminal to the Lys-C cleavage site.

Gbeta subunits have been shown to mediate GRK2 and GRK3 (betaARK1 and betaARK2) translocation to retinal ROS containing the light receptor substrate rhodopsin, cell membranes (Pitcher et al. 1992), or to phospholipid vesicles (Kim et al., 1993). In addition, they robustly stimulate the activity of the kinase 10-20-fold toward receptor substrates but much less so toward synthetic peptide substrates (Pitcher et al. 1992). Cleavage of the Gbeta- complex with Lys-C retained the two fragments 1-32(36) and 33(37)-68(71) non-covalently associated with the Gbeta subunit. Tryptic fragments of the Gbeta complex cleaved within Gbeta similarly remain attached (Fung and Nash, 1983; Thomas et al., 1993). G is not cleaved by trypsin in the Gbeta complex (Tamir et al., 1991). The Lys-C Gbeta fragment complex retained the ability to function as a binding site for the PH domain of betaARK1, to productively associate with Galpha(o)/Galpha(i) to allow those proteins to serve as pertussis toxin substrates, and to inhibit Type I adenylate cyclase activity. On the other hand, the cleaved complex only weakly stimulated betaARK1 phosphorylation of rhodopsin. Lys-C cleavage may not be unique in diminishing Gbeta stimulation of rhodopsin phosphorylation by betaARK1. The effect of trypsin cleavage of Gbeta has not been assayed.

The inability of N-terminal Gbeta or G peptides or immunoprecipitating antibodies to these peptides to affect Gbeta-dependent rhodopsin phosphorylation or binding to the PH domain suggests that a region of the Gbeta other than the N terminus is involved in these interactions. This suggestion is consistent with the findings of Wang et al.(1994) that betaARK PH domain binding may be mediated through the C-terminal five WD40 motifs of Gbeta rather than through G.

There may be multiple points of interaction between Gbeta, betaARK, and other molecules. Some regions may specify binding to target molecules while others may modulate the catalytic activity of betaARK in conjunction with the ligand-activated receptor substrate. The modulation of betaARK activity could also be occurring at the level of Gbeta interaction with the receptor substrate. While the fragments of Lys-C-digested G appear to remain non-covalently associated in solution as the Gbeta dimer, either they are displaced in the receptor complex with Gbeta, or they fail to assume the appropriate relationship to the rest of the members of the complex to activate betaARK phosphorylation of the receptor. Nicking of the G subunit by proteolysis with Lys-C indicates that the Gbeta dimer provides active modulation rather than a passive kinase binding scaffold for the phosphorylation of G-protein-coupled receptor substrates by betaARK1.


FOOTNOTES

*
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. Tel.: 313-998-5929; Fax: 313-996-5668; :levineh{at}aa.wl.com.

(^1)
The abbreviations used are: GRK, G-protein receptor kinase; betaARK, beta-adrenergic receptor kinase; PH, pleckstrin homology; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycine; ROS, rod outer segments; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Tracy Stevenson and Margie Whitton in Analytical Chemistry at Parke-Davis for MALDI mass spectroscopy and for determining the amino acid sequence of the isolated G subunits, respectively. Dr. Richard Neubig and Sue Wade of the Department of Pharmacology at the University of Michigan Medical School graciously provided platelet membranes for the adenylate cyclase assays and Gbeta(2)(3-29) and G(2)(8-34) synthetic peptides.


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