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• W2002341497 abstract "Cleavage after lysine 32 in the Gγ2 subtype and after lysine 36 in the Gγ3 subtype of purified mixed brain Gβγ by endoproteinase Lys-C blocks Gβγ-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human β-adrenergic receptor kinase (hβARK1) holoenzyme while hβARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gβγ to C-terminal fragments of hβARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gβγ with phospholipids, its ability to support pertussis toxin-catalyzed Gαo/Gαi ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gβ subunit remains noncovalently associated with the cleaved Gγ fragments. Thus, in addition to recruiting hβARK1 to its receptor substrate, Gγ contributes secondary and/or tertiary structural features to activate the kinase. Cleavage after lysine 32 in the Gγ2 subtype and after lysine 36 in the Gγ3 subtype of purified mixed brain Gβγ by endoproteinase Lys-C blocks Gβγ-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human β-adrenergic receptor kinase (hβARK1) holoenzyme while hβARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gβγ to C-terminal fragments of hβARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gβγ with phospholipids, its ability to support pertussis toxin-catalyzed Gαo/Gαi ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gβ subunit remains noncovalently associated with the cleaved Gγ fragments. Thus, in addition to recruiting hβARK1 to its receptor substrate, Gγ contributes secondary and/or tertiary structural features to activate the kinase. INTRODUCTIONG-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), ( 1The abbreviations used are: GRKG-protein receptor kinaseβARKβ-adrenergic receptor kinasePHpleckstrin homologyPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferaseTricineN-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycineROSrod outer segmentsHPLChigh performance liquid chromatography.) 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 (βARK1 and βARK2) subfamilies are encoded by separate genes (Benovic et al., 1991). They were originally shown to phosphorylate the β2-adrenergic receptor and thus were given the name β-adrenergic receptor kinases or βARKs. The βARKs are C-terminally extended relative to other members of the GRK family. The C-terminal 222 amino acids of βARK1 and βARK2 contain a domain responsible for the association of the enzyme with heterotrimeric G-protein Gβγ subunits (Pitcher et al., 1992). Addition of Gβγ subunits to an in vitro phosphorylation system stimulates βARK 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 (5er et al., 1993). While the portions of the βARK C terminus involved in the association with Gβγ subunits have been delineated (Koch et al., 1993), less is known about the determinants on the Gβγ partner. The multiple subtypes of Gβ and Gγ, the requirement for the Gβγ 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 Gβγ binding to hβARK1 from Gβγ stimulation of rhodopsin phosphorylation by this kinase after proteolysis with Lys-C. Gγ is cleaved by the protease at lysine 33 in Gγ2 (lysine 36 in Gγ3), and the Gγ fragments remain associated with the Gβ subunit.EXPERIMENTAL PROCEDURESFrozen bovine retinas were from George A. Hormel, Austin, MN. Frozen bovine brain was from Pel-Freez, Rogers, AR. ATP, GDP, GTPγS, 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 β1, β2, β3, β2, pan-β, γ2, γ3, γ4, and γ5 of the heterotrimeric G-protein Gβγ 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 βARK1 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 βARK1, eluting with 0.1 M glycine, pH 2. Secondary antibody (donkey anti-rabbit peroxidase) and ECL detection reagents were purchased from Amersham. [γ-32P]ATP (3000 Ci/mmol), [γ-35S]GTP (1045 Ci/mmol), and [α-32P]NAD+ (30 Ci/mmol) were from DuPont NEN. S-Adenosyl-L-[3H-methyl]methionine (77 Ci/mmol), the 125I-cAMP Scintillation Proximity Assay Kit, Rainbow prestained molecular weight markers, and Amplify were acquired from Amersham. Purified recombinant human βARK His6 (PH+C) (Gly556-Ser670) protein was generously provided by Dr. Daruka Mahadevan (Mahadevan et al., 1995). DNA encoding the βARK C-terminal domains βARK (PH+C) (Gly556-Ser670) and the C-terminal 222 amino acids (Pro466-Leu689) were cloned by PCR from the human βARK1 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 Gγ2 or residues 3-29 of Gβ2 were kindly provided by Dr. R. Neubig, Department of Pharmacology, University of Michigan.Heterotrimeric G-proteins were isolated from frozen bovine brain, and the Gβγ subunit complex (mixed subtypes of β and γ 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 Gβγ from Gα 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 Gα and Gβγ subunits were stored in aliquots at −80°C. Two different batches of Gβγ when assayed for effects of Lys-C proteolysis on rhodopsin phosphorylation and hβARK1 binding activity revealed no discernible differences. Carboxymethylation of the C terminus of the γ subunit of Gβγ 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 32P-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 32P 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. βARK1 and Gβ 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 hβARK1 recognizing the PH domain of that kinase (Sallese et al., 1995) was used to detect the enzyme. Gβ subunits were detected with pan-β subunit-specific antibodies. β and γ 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 βARK1 and were detected with Gβ- or Gγ- subtype subunit-specific antibodies. Quantitation of the ECL was standardized as described (Mahadevan et al., 1995).Measurement of the Ability of Gβγ to Stimulate Rhodopsin or Synthetic Peptide Phosphorylation by hβARK1Gβγ 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 MgCl2, 16 μCi/ml [γ-32P]ATP, 50 μM ATP, 10 μM GTPγS, and 160 pmol of rhodopsin as urea-extracted ROS. After 10 min on ice, 5 μl of 50 μg/ml recombinant hβARK1 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 hβARK1 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 hβARK1, and absence or presence of 178 nM Gβγ under a fluorescent table lamp at room temperature in 20 mM Tris-HCl, pH 7.8, 2 mM EDTA, 10 mM MgCl2, 2 mM dithiothreitol. The ROS were pelleted at 4°C at 109,000 × 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.Gβγ Subunit-mediated ADP-ribosylation of Gαo and Gαi by Pertussis ToxinThe ability of Gβγ subunits to support pertussis toxin-catalyzed [32P]ADP-ribose transfer from [α-32P]NAD+ to Gαo/Gαi 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 MgCl2, 1 mM EDTA, and 10 μM GDP. The dose response for Gβγ subunits was determined at a constant Gαo/Gαi concentration. After SDS-PAGE, the incorporation of 32P into polypeptides migrating in the Gα region was determined by densitometry from autoradiograms.Adenylate Cyclase Inhibition by Gβγ SubunitsDose-dependent inhibition of platelet membrane type I adenylate cyclase by Gβγ 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 Gβγ 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 MgCl2, 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 Gβγ 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 × 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 Gβγ by Endoproteinase Lys-CCleavage of the Gβγ 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 Gβγ 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 Gβγ at 30°C without protease had no discernible effect on its measured biochemical properties.Binding of Gβγ Subunits to PH Domain-containing C-terminal βARK1 FragmentsGβγ subunit binding to immobilized PH domains was determined by immunoblot analysis with pan-anti-β subunit antibodies as described by Mahadevan et al.(1995).Amino Acid Sequencing of Lys-C-cleaved GβγSeparation of the Gβ and Gγ subunits was achieved by C4 reverse phase chromatography under the conditions used to separate the farnesylated γ subunit of transducin on a C18 column (Parish and Rando, 1994). Western blot analysis showed that the Gβ and Gγ subunits were separated under these conditions (data not shown). Fifty μg of Lys-C-treated Gβγ (~1.2 nmol) was diluted with an equal volume of 6 M guanidine HCl, 50 mM dithiothreitol and injected onto a Brownlee 4.6 × 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 γ subunits as judged by comigration of digested [3H]carboxymethylated Gβγ (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.RESULTSThe Gβγ subunits of the heterotrimeric G-proteins form a heterodimer (β = 35 kDa or 36 kDa, γ= 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 Gα 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 Gγs 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 Gβγ 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). Gβγ requires small amounts of detergent to remain in solution.Lys-C Proteolysis of GβγThe structure of the Gβγ 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 Gβγ.Reducing SDS-PAGE analysis of the proteolytic product as a function of protease:Gβγ ratio revealed a size range for the Gγ fragment of Mr 3400-5200 by silver staining, a reduction from Mr 6900-8600, corresponding to an Mr of 3500. There was no discernible effect on the Mr of the Gβ subunit (Fig. 1). The mixed Gβγ preparation isolated from frozen bovine brain contains multiple subtypes of Gβ and Gγ subunits, the latter accounting for the smear around Mr 6500. Antibodies raised to synthetic peptides corresponding to the N-terminal 17 residues of the Gγ subunits or 21 amino acids of the Gβ 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 Gβγ used in these studies has the following distribution of Gβ and Gγ subunits: β4≫β2 > β1 > β3; γ2≫γ3≫γ2 > 5 (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 Gβγ subunits could influence the recovery and thus the apparent distribution of the different subtypes.Methylation of the C-terminal cysteine (Cys68) carboxyl moiety of the purified brain Gβγ with S-adenosyl-L-[3H-methyl]methionine (Fung et al., 1990) provided a marker for the C terminus of the Gγ subunit. Lys-C treatment of the tritiated Gβγ released a labeled fragment of Mr 3500 (Fig. 2), mirroring the shift on SDS-PAGE to lower Mr seen with silver staining (Fig. 1). This implies that the extreme C terminus of the Gγ subunit remains intact. Immunoblotting after SDS-PAGE showed that N-terminal Gγ subunit immunoreactivity (residues 2-17) was eliminated by Lys-C treatment (Fig. 3). By contrast, for the Gβ 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 Gβγ on a Superose 12 column in 0.8% cholate before or after Lys-C treatment demonstrated that the fragments of the cleaved Gγ subunit remained complexed with the Gβ 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 Gβγ was observed. By contrast, denaturing SDS-PAGE and subsequent Western blot analysis of the same fractions revealed a significant loss of N-terminal Gγ subunit immunoreactivity (Table 1).Figure 2:Reduction in Mr of C-terminal [3H]carboxymethylated bovine brain Gβγ by Lys-C proteolysis visualized by 3H autoradiography. 100 μg of bovine brain Gβγ 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 Mr 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 Mr~3500 decrease in size of the labeled Gγ subunit. The leading ion front ran to the very bottom of the gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Removal of N-terminal Gγ subunit immunoreactivity by Lys-C proteolysis. A total of 10 μg of bovine brain Gβγ 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 Gγ-antibodies specific for the N-terminal residues(2.Benovic J.L. Onorato J.J. Arriza J.L. Stone W.C. Lohse M. Jenkins N.A. Gilbert D.J. Copeland N.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1991; 266: 14939-14946Abstract Full Text PDF PubMed Google Scholar, 3.Bubus J. Khorana H.G. J. Biol. Chem. 1990; 265: 12995-12999Abstract Full Text PDF PubMed Google Scholar, 4.Calbiochem A Guide to the Properties and Uses of Detergents in Biology and Biochemistry. Calbiochem-Novabiochem Corp., San Diego1993Google Scholar, 5.Chuang T.T. Sallese M. Ambrosini G. Parruti G. De Blasi A. J. Biol. Chem. 1992; 267: 6886-6892Abstract Full Text PDF PubMed Google Scholar, 6.Clapham D.E. Neer E.J. Nature. 1993; 365: 403-406Crossref PubMed Scopus (588) Google Scholar, 7.Cook P.F. Nelville Jr., M.E. Vrana K.E. Hartl F.T. Roskoski Jr., R. Biochemistry. 1982; 21: 5794-5799Crossref PubMed Scopus (346) Google Scholar, 8.Fung B.K. Nash C.R. J. Biol. Chem. 1983; 258: 10503-10510Abstract Full Text PDF PubMed Google Scholar, 9.Fung K.-K. Yamane H.K. Ota I.M. Clarke S. FEBS Lett. 1990; 260: 313-317Crossref PubMed Scopus (9) Google Scholar, 10.Garritsen A. Simonds W.F. J. Biol. Chem. 1994; 269: 24418-24423Abstract Full Text PDF PubMed Google Scholar, 11.Gibson T.J. Hyvonen M. Musacchio A. Saraste M. Trends Biochem. Sci. 1994; 19: 349-353Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 12.Haga K. Haga T. FEBS Lett. 1990; 268: 43-47Crossref PubMed Scopus (57) Google Scholar, 13.Haga K. Haga T. J. Biol. Chem. 1992; 267: 2222-2227Abstract Full Text PDF PubMed Google Scholar, 14.Huff R.M. Neer E.J. J. Biol. Chem. 1986; 261: 1105-1110Abstract Full Text PDF PubMed Google Scholar, 15.Inglese J. Freedman N.J. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 23735-23738Abstract Full Text PDF PubMed Google Scholar, 16.Ingley E. Hemmings B.A. J. Cell. Biochem. 1994; 56: 436-443Crossref PubMed Scopus (67) Google Scholar, 17.Iniguez-Lluhi J.A. Simon M.I. Robishaw J.D. Gilman A.G. J. Biol. Chem. 1992; 267: 23409-23417Abstract Full Text PDF PubMed Google Scholar) of the Gγ2 and Gγ3 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:Gβγ (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Amino Acid Sequence of the Lys-C-treated Gγ SubunitGβγ 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. 4 shows the migration of the [3H]carboxymethyl marker of the C terminus of the Gγ subunit on a separate HPLC run. The arrow marks the position of the truncated C-terminal Gγ sequences (Ala33-Cys67 for Gγ2; Ala37-Cys67 for Gγ3). The two Gγ 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 Gγ2 and Gγ3 polypeptides, while the cluster of radioactive peaks around fraction 40 represent undissociated Gβγ and the unidentified carboxymethylated Mr 24,000 protein in brain membranes (see Fig. 1 and Fung et al.(1990)). Table 2 shows the sequences obtained from the major peaks of 214 nm absorbance produced by Lys-C treatment of unlabeled isolated Gβγ subunits. Gγ2, the major immunoreactive Gγ subtype in the purified brain Gβγ preparation, was likewise the prominent proteolytic fragment represented. Both Gγ2 and Gγ3 yielded peptides beginning with Ala33 (numbering system of Gγ2, A36 of Gγ3) immediately following Lys32, 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 Mr change in Gγ seen by SDS-PAGE (~3500, Figure 1:, Figure 2:, Figure 3:). Gγ sequences corresponding to other potential Lys-C proteolytic fragments of Gγ2, Gγ3, or other known Gγ subtypes were not detected. Minor 214 nm absorbance peaks contained short C-terminal Gγ 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 Gγ2 and Gγ3 were also detected. The same sequencing results were obtained for two independent preparations of Gβγ subunits.Figure 4:C4 reverse phase chromatography of [3H]carboxymethylated Gβγ subunits. Fifteen μg of [3H]carboxymethylated Gβγ 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 Gγ peptides (see text); asterisk, undigested Gγ2, G γ3 polypeptides.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Functional Effects of Cleavage of the Gγ Subunit: Biochemical Activities of Gβγ SubunitsSince Gβγ 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 Gβγ subunits was evaluated. Their ability to bind to ROS and to modulate rhodopsin phosphorylation by full-length recombinant hβARK1 was measured. Their ability to bind to the GST-βARK C-terminal domain of 222 amino acids (Pro466-Leu689) and to bind to a more restricted region of the kinase, GST- or His6-tagged βARK PH+C domain (βARK G556-S670), was determined. Finally, the ability of the treated and control Gβγ subunits to associate with and to modulate Gα subunit activities and to regulate the activity of an effector, Type I adenylate cyclase, were also assessed.Modulation of βARK1-mediated Receptor PhosphorylationThe Lys-C-treated Gβγ subunit preparation supported substantial translocation of human βARK1 to isolated retinal rod outer segments (Fig. 5A), but failed to stimulate phosphorylation of rhodopsin (Fig. 5B). Gβγ subunits treated with leupeptin-inhibited Lys-C fully supported both translocation and phosphorylation. Fig. 5C shows that Lys-C proteolysis of Gγ2 and Gγ3 abrogates stimulation of βARK1 phosphorylation of receptor substrates without abolishing binding of the kinase. The" @default.
• W2002341497 created "2016-06-24" @default.
• W2002341497 creator A5004043162 @default.
• W2002341497 creator A5022285559 @default.
• W2002341497 creator A5056201594 @default.
• W2002341497 date "1996-02-01" @default.
• W2002341497 modified "2023-10-01" @default.
• W2002341497 title "An Intact N Terminus of the γ Subunit Is Required for the Gβγ Stimulation of Rhodopsin Phosphorylation by Human β-Adrenergic Receptor Kinase-1 but Not for Kinase Binding" @default.
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