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• W2021008077 abstract "Catalysis of nucleotide exchange in heterotrimeric G proteins (Gαβγ) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Gα N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R*) interaction. The N-terminal fatty acylation of Gα is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Gα. In Gαβγ, lack of the fatty acid and Gα truncations up to 33 amino acids had little effect on R* binding and R*-catalyzed nucleotide exchange, implying that this region is not mandatory for R*/Gαβγ interaction. However, when the other hydrophobic modification of Gαβγ, the Gγ C-terminal farnesyl moiety, is lacking, R* interaction requires the fatty acylated Gα N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Gαβγ with R*. We propose that in native Gαβγ, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R*. Accordingly, we find that the native fatty acylated Gα is competent to interact with R* even in the absence of Gβγ, whereas nonacylated Gα requires Gβγ for interaction. Experiments with N-terminally truncated Gα subunits suggest that in the second step of the catalytic process, the receptor binds to the αN/β1-loop region of Gα to reduce nucleotide affinity and to make the Gα C terminus available for subsequent interaction with R*. Catalysis of nucleotide exchange in heterotrimeric G proteins (Gαβγ) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Gα N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R*) interaction. The N-terminal fatty acylation of Gα is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Gα. In Gαβγ, lack of the fatty acid and Gα truncations up to 33 amino acids had little effect on R* binding and R*-catalyzed nucleotide exchange, implying that this region is not mandatory for R*/Gαβγ interaction. However, when the other hydrophobic modification of Gαβγ, the Gγ C-terminal farnesyl moiety, is lacking, R* interaction requires the fatty acylated Gα N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Gαβγ with R*. We propose that in native Gαβγ, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R*. Accordingly, we find that the native fatty acylated Gα is competent to interact with R* even in the absence of Gβγ, whereas nonacylated Gα requires Gβγ for interaction. Experiments with N-terminally truncated Gα subunits suggest that in the second step of the catalytic process, the receptor binds to the αN/β1-loop region of Gα to reduce nucleotide affinity and to make the Gα C terminus available for subsequent interaction with R*. Signal transduction by heterotrimeric G proteins (Gαβγ) and G protein-coupled receptors (GPCRs) 4The abbreviations used are: GPCR, G protein-coupled receptor; Gt, G protein of the rod cell, transducin; CTα, C-terminal tail of Gα;CTγ, (farnesylated) C-terminal tail of Gγ;NTα, (myristoylated) N-terminal region of Gα; MII, metarhodopsin II; R*, active state of rhodopsin; GTPγS, guanosine 5′-O-(thiotriphosphate). 4The abbreviations used are: GPCR, G protein-coupled receptor; Gt, G protein of the rod cell, transducin; CTα, C-terminal tail of Gα;CTγ, (farnesylated) C-terminal tail of Gγ;NTα, (myristoylated) N-terminal region of Gα; MII, metarhodopsin II; R*, active state of rhodopsin; GTPγS, guanosine 5′-O-(thiotriphosphate). is a fundamental process in the regulation of cellular function (1Lu Z.L. Saldanha J.W. Hulme E.C. Trends Pharmacol. Sci. 2002; 23: 140-146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 2Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (508) Google Scholar). A prototypical GPCR and the eponym of the largest family of the GPCR superfamily is rhodopsin, the visual pigment of retinal rod photoreceptor cells. Photon capture by rhodopsin's covalently bound chromophore 11-cis-retinal causes retinal cis/trans isomerization and thus activates rhodopsin. Within milliseconds, subsequent conversions in the protein moiety lead to the active rhodopsin conformation (R*), which is capable of catalyzing GDP/GTP exchange in the retinal G protein transducin (Gt) (3Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 4Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2003; 65: 851-879Crossref PubMed Scopus (195) Google Scholar). In the inactive state, Gt consists of Gαt·GDP and the Gβ1γ1 dimer (5Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar) (see Fig. 1A).A great deal of data regarding rhodopsin and Gt are available: high resolution structures of both rhodopsin (6Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4991) Google Scholar, 7Okada T. Sugihara M. Bondar A.N. Elstner M. Entel P. Buss V. J. Mol. Biol. 2004; 342: 571-583Crossref PubMed Scopus (929) Google Scholar, 8Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (669) Google Scholar) and Gt (5Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar, 9Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (702) Google Scholar, 10Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (521) Google Scholar); information on interaction sites on receptor and G protein (reviewed in Refs. 2Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (508) Google Scholar, 4Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2003; 65: 851-879Crossref PubMed Scopus (195) Google Scholar, and 11Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4819-4821Crossref PubMed Scopus (178) Google Scholar); experimental (e.g. see Refs. 2Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (508) Google Scholar and 12Rondard P. Iiri T. Srinivasan S. Meng E. Fujita T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6150-6155Crossref PubMed Scopus (78) Google Scholar, 13Natochin M. Moussaif M. Artemyev N.O. J. Neurochem. 2001; 77: 202-210Crossref PubMed Scopus (37) Google Scholar, 14Marin E.P. Krishna A.G. Sakmar T.P. Biochemistry. 2002; 41: 6988-6994Crossref PubMed Scopus (49) Google Scholar, 15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Abdulaev N.G. Ngo T. Zhang C. Dinh A. Brabazon D.M. Ridge K.D. Marino J.P. J. Biol. Chem. 2005; 280: 38071-38080Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and theoretical studies (17Filipek S. Krzysko K.A. Fotiadis D. Liang Y. Saperstein D.A. Engel A. Palczewski K. Photochem. Photobiol. Sci. 2004; 3: 628-638Crossref PubMed Scopus (153) Google Scholar, 18Fanelli F. De Benedetti P.G. Chem. Rev. 2005; 105: 3297-3351Crossref PubMed Scopus (142) Google Scholar); and various concepts of R*/Gt interaction (11Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4819-4821Crossref PubMed Scopus (178) Google Scholar, 15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 19Kisselev O. Pronin A. Ermolaeva M. Gautam N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9102-9106Crossref PubMed Scopus (96) Google Scholar, 20Bohm A. Gaudet R. Sigler P.B. Curr. Opin. Biotechnol. 1997; 8: 480-487Crossref PubMed Scopus (77) Google Scholar, 21Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar, 22Iiri T. Farfel Z. Bourne H.R. Nature. 1998; 394: 35-38Crossref PubMed Scopus (162) Google Scholar, 23Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar, 24Yeagle P.L. Albert A.D. Biochemistry. 2003; 42: 1365-1368Crossref PubMed Scopus (44) Google Scholar, 25Cherfils J. Chabre M. Trends Biochem. Sci. 2003; 28: 13-17Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Chabre M. le Maire M. Biochemistry. 2005; 44: 9395-9403Crossref PubMed Scopus (190) Google Scholar, 27Shichida Y. Morizumi T. Photochem. Photobiol. 2006; (in press)PubMed Google Scholar, 28Oldham W. Van Eps N. Preininger A. Hubbell W.L. Hamm H.E. Nat. Struct. Mol. Biol. 2006; (in press)PubMed Google Scholar). Despite the abundance of this information, however, it has not been sufficient to comprehensively describe how R* catalyzes nucleotide exchange in the G protein. The side of Gt facing R* (side oriented downward in Fig. 1A) (29Zhang Z. Melia T.J. He F. Yuan C. McGough A. Schmid M.F. Wensel T.G. J. Biol. Chem. 2004; 279: 33937-33945Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) contains two key interaction sites, the C-terminal tail of Gαt (CTα) and the C-terminal tail of Gγ1 with its farnesyl modification (CTγ). CTα and farnesylated CTγ selectively recognize R* (30Kisselev O.G. Meyer C.K. Heck M. Ernst O.P. Hofmann K.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4898-4903Crossref PubMed Scopus (104) Google Scholar, 31Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (31) Google Scholar) and adopt helical conformations upon binding (32Kisselev O.G. Kao J. Ponder J.W. Fann Y.C. Gautam N. Marshall G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4270-4275Crossref PubMed Scopus (163) Google Scholar, 33Koenig B.W. Kontaxis G. Mitchell D.C. Louis J.M. Litman B.J. Bax A. J. Mol. Biol. 2002; 322: 441-461Crossref PubMed Scopus (94) Google Scholar, 34Kisselev O.G. Downs M.A. Structure (Camb.). 2003; 11: 367-373Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Involvement of the fatty acylated N-terminal region of Gαt (NTα; see Fig. 1A) in catalytic R*/G protein interaction was suggested by a study on a form of Gαβγ lacking a farnesyl moiety (15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). There, fatty acylation of the Gα N terminus enabled interaction with R*. This and other results (35Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H.R. Science. 1997; 275: 381-384Crossref PubMed Scopus (196) Google Scholar, 36Itoh Y. Cai K. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4883-4887Crossref PubMed Scopus (102) Google Scholar, 37Blahos J. Fischer T. Brabet I. Stauffer D. Rovelli G. Bockaert J. Pin J.P. J. Biol. Chem. 2001; 276: 3262-3269Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 38Taylor J.M. Jacob-Mosier G.G. Lawton R.G. Remmers A.E. Neubig R.R. J. Biol. Chem. 1994; 269: 27618-27624Abstract Full Text PDF PubMed Google Scholar) indicate the presence of NTα in the R*/Gt interface, although its function there is not well understood.In this work, we set out to elucidate the role of NTα in G protein/receptor coupling by investigating N-terminal truncated Gα subunits. We confirm that lack of NTα has an effect on subunit interaction with Gβγ (39Navon S.E. Fung B.K. J. Biol. Chem. 1987; 262: 15746-15751Abstract Full Text PDF PubMed Google Scholar, 40Graf R. Mattera R. Codina J. Estes M.K. Birnbaumer L. J. Biol. Chem. 1992; 267: 24307-24314Abstract Full Text PDF PubMed Google Scholar, 41Nanoff C. Koppensteiner R. Yang Q. Fuerst E. Ahorn H. Freissmuth M. Mol. Pharmacol. 2006; 69: 397-405Crossref PubMed Scopus (33) Google Scholar). We show that the G protein's two terminal regions carrying hydrophobic modifications (i.e. myristoylated NTα and farnesylated CTγ) are functionally redundant parts of an amphiphilic microdomain that serves to anchor the G protein to the membrane and affords collisional coupling with R*. The actual trigger of nucleotide exchange requires a subsequent step that makes the Gα C terminus available for interaction with cytoplasmic binding sites of R* (15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).EXPERIMENTAL PROCEDURESMaterials—Rod outer segments and disk membranes were prepared from frozen dark-adapted retinas as described (42Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Gt heterotrimers were purified from rod outer segment preparations essentially as described previously (42Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 43Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Crossref PubMed Google Scholar). Subsequent separation into the Gαt subunit and the Gβγ dimer was performed by chromatography on a HiTrap Blue column (GE Healthcare Life Sciences) as described (42Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The contamination of Gαt preparations by Gβγ was estimated to be less than 1%. This result was based on evaluation of the initial rates of R*-induced GTPγS uptake by Gαt with decreasing Gβγ concentrations using the Gt fluorescence activation assay.Peptide Synthesis—Peptide synthesis, myristoylation, and purification were as described previously (44Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Lyophilized peptides were dissolved in water, and the pH was adjusted with 1 m NaOH to pH 6. Peptides were either unmodified or myristoylated at the N-terminal Gly.UV-visible Spectroscopy—The amount of “extra MII” was recorded by time-resolved UV-visible spectroscopy as described (45Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Crossref PubMed Google Scholar). Samples contained 10 μm rhodopsin in disk membranes. Measurements were performed at pH 8.0 and 4 °C. Cuvette path length was 2 mm. 11.5% of rhodopsin was flash-activated by 500 ± 20-nm light.Near Infrared Kinetic Light Scattering—Changes in intensities of scattered light were measured as described (43Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Crossref PubMed Google Scholar). All measurements were performed in 10-mm path cuvettes at pH 7.4 and 23 ± 1 °C. Reactions were triggered by flash photolysis of rhodopsin (3 μm) in disk membranes using flashes of green light (500 ± 20 nm; the flash activated 32% of rhodopsin).Transducin Fluorescence Activation Assay—Gt activation was monitored by relative changes in intrinsic fluorescence emission at 340 nm after excitation at 300 nm as described before (45Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Crossref PubMed Google Scholar). Samples containing rhodopsin in disk membranes (50 nm), Gα (0.6 μm), and Gβγ (0.5 μm) were illuminated with orange light (>495 nm, 10 s). Nucleotide uptake by the G protein was induced by adding GTPγS(5 μm final concentration) at 20 °C and constant stirring. The intrinsic GDP/GTP exchange of Gα subunits (0.6 μm) was monitored in the absence of Gβγ and rhodopsin (see Ref. 45Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Crossref PubMed Google Scholar and references therein). To complete activation of the entire Gα pool, NaF and AlCl3 were added sequentially with ∼20 s between additions (final concentrations: 3.7 mm and 100 μm, respectively). Traces were normalized to the final fluorescence levels evoked by the Gα·GTPγS/Gα·GDP·AlF-4 mixture (15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).Size Exclusion Chromatography—Size exclusion chromatography was used to characterize the G protein subunit association in solution using the molecular weight shift that occurs upon heterotrimer formation. The subunits (∼6 μm, 50 μl final volume) were loaded on a Superose 12 column (0.32 × 30 cm; GE Healthcare Life Sciences) equilibrated with 20 mm, 1,3-bis-(tris(hydroxymethyl)-methyl-amino)propane (BTP), pH 7.5, 130 mm NaCl, 1 mm MgCl2, 2 mm dithiothreitol (10 °C) at a flow rate of 40 μl/min using a Smart System (GE Healthcare Life Sciences). The elution was monitored at 280 nm, and 40 μl fractions were collected for subsequent SDS-PAGE analysis.Cloning, Expression, and Purification of Gα Subunits—Expression of non-fatty acylated Gα subunits in Escherichia coli and purification were as described before (15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Experiments were performed with rat Gαi1 (termed Gαi) and a Gαt/Gαi1 chimera (here termed rGαt; originally termed Gtα* in Ref. 46Natochin M. Granovsky A.E. Muradov K.G. Artemyev N.O. J. Biol. Chem. 1999; 274: 7865-7869Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), which contains 16 residues from Gαi1 and can be expressed in E. coli (46Natochin M. Granovsky A.E. Muradov K.G. Artemyev N.O. J. Biol. Chem. 1999; 274: 7865-7869Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The expression vector pHis6-Gtα* for expression of rGαt was kindly provided by M. Natochin and N. Artemyev. Both Gα subunits are known to couple in combination with Gβ1γ1, as transducin couples to activated rhodopsin (47Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 48Natochin M. Granovsky A.E. Artemyev N.O. J. Biol. Chem. 1998; 273: 21808-21815Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In order to produce Δ25-rGαt, bases corresponding to residues 2-25 of the Gαt sequence were deleted by a PCR using pHis6-Gtα* as a template. The PCR product was digested with NcoI and BstEII and subcloned into pHis6-Gtα*. For construction of Gαi mutants with N-terminal deletions, oligonucleotide duplexes with respective deletions were cloned into the EcoRI and BssHII restriction sites of the plasmid pGiL, which harbors the sequence coding for rat Gαi1 with an internal His6 tag (15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The subunits termed Gαi-t, Δ29-Gαi-t, Δ31-Gαi-t, Δ33-Gαi-t, and Δ35-Gαi-t are derivatives of Gαi (vector pGiL) with a single Asn → Glu amino acid exchange to yield the Gαt C-terminal sequence (Fig. 1B). For Gαi-t and the respective deletion mutants, we obtained about 3 mg of pure protein/liter of cell culture.RESULTSIntrinsic Nucleotide Exchange and Heterotrimer Formation of Gα Subunits with N-terminal Deletions—In order to study the role of NTα in catalytic R*/G protein interaction and subunit interaction with Gβγ, we expressed three sets of Gα subunits in E. coli. Each set was composed of full-length Gα and mutants with N-terminal truncations of various length (see Fig. 1B). The most extensive truncation included the end of the N-terminal helix and first residues of the β1 strand, which were reported to be involved in receptor contact (36Itoh Y. Cai K. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4883-4887Crossref PubMed Scopus (102) Google Scholar, 37Blahos J. Fischer T. Brabet I. Stauffer D. Rovelli G. Bockaert J. Pin J.P. J. Biol. Chem. 2001; 276: 3262-3269Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Since light-activated rhodopsin can interact with both Gαt/Gβ1γ1 and Gαi/Gβ1γ1 (47Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 48Natochin M. Granovsky A.E. Artemyev N.O. J. Biol. Chem. 1998; 273: 21808-21815Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), the first set was derived from recombinant Gαt (rGαt and Δ25-rGαt). rGαt is a chimera containing 16 residues from Gαi, which allows functional expression in E. coli (46Natochin M. Granovsky A.E. Muradov K.G. Artemyev N.O. J. Biol. Chem. 1999; 274: 7865-7869Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The other two sets were based on Gαi (Gαi and Δ29-Gαi) and a Gαi mutant containing the N346E substitution, which changes the C-terminal tail to the corresponding Gαt sequence (Gαi-t, Δ29-Gαi-t, Δ31-Gαi-t, Δ33-Gαi-t, and Δ35-Gαi-t). Gαi-t was preferentially used in the study, because the protein's termini are not modified by an affinity tag, and interaction with R* is similar to Gαt (11Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4819-4821Crossref PubMed Scopus (178) Google Scholar, 47Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 48Natochin M. Granovsky A.E. Artemyev N.O. J. Biol. Chem. 1998; 273: 21808-21815Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar).We measured intrinsic GTPγS-induced fluorescence increase of isolated Gα subunits to study the influence of the different N-terminal truncations on intrinsic nucleotide exchange. Almost the same rates of GTPγS uptake were found for Gαi and Δ29-Gαi, in agreement with published data (41Nanoff C. Koppensteiner R. Yang Q. Fuerst E. Ahorn H. Freissmuth M. Mol. Pharmacol. 2006; 69: 397-405Crossref PubMed Scopus (33) Google Scholar) (data not shown). This was also observed for Gαi-t, Δ29-Gαi-t, and Δ31-Gαi-t, whereas Δ33-Gαi-t showed a slightly faster GTPγS uptake (Fig. 2A). After the addition of AlF-4, all of these subunits formed Gα·GDP·AlF-4 complexes, thus demonstrating functional folding of the Gα subunits. However, Δ35-Gαi-t did not show any fluorescence increase, after the addition of either GTPγSorAlF-4 (Fig. 2A, bottom trace), indicating that no GDP was bound in the nucleotide binding pocket.FIGURE 2Intrinsic nucleotide exchange and heterotrimer formation of N-terminal truncated Gα subunits. A, intrinsic nucleotide exchange of the Gα subunits (0.6 μm) Gαi-t (black), Δ29-Gαi-t (blue), Δ31-Gαi-t (green), Δ33-Gαi-t (red), and Δ35-Gαi-t (gray) was monitored by measuring the increase in fluorescence emission after the addition of GTPγS (5 μm; black arrow). The remaining Gα·GDP was activated by the addition (gray arrows) of NaF and AlCl3 to form Gα·GDP·AlF-4. Traces were normalized to the final fluorescence intensity. Nonnormalized final fluorescence intensities of different Gα subunits varied within 15%. B, heterotrimer formation of Gαi-t, Δ29-Gαi-t, Δ31-Gαi-t, and Δ33-Gαi-t with Gβγ investigated by size exclusion chromatography (upper panel). Elution profiles are shown for Gα alone (black), Gβγ alone (red), and the mixture of Gα/Gβγ (green). Calculated superpositions of the single Gα and Gβγ elution profiles are shown as dotted lines. Lower panel, SDS-PAGE analysis of the elution fractions. Note that in the right panel the Gα and Gβ bands are close to each other due to the weaker separation performance of the SDS gels used.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We used size exclusion chromatography to study heterotrimer formation of truncated nonacylated Gα subunits with Gβγ. Nonacylated Gαi-t, Δ29-Gαi-t, Δ31-Gαi-t, and Δ33-Gαi-t as well as Gβγ eluted as single peaks from the size exclusion column, demonstrating the homogeneity of the subunit preparations (Fig. 2B). When combined with Gβγ, all four Gα subunits showed single peak elution profiles and shorter retention times as compared with the isolated Gα subunits. Almost identical results were obtained with nonacylated Gαi in combination with Gβγ (data not shown). Although truncation of the N-terminal region reduces the affinity of the subunits as previously reported for Gαt and other Gα subunits (39Navon S.E. Fung B.K. J. Biol. Chem. 1987; 262: 15746-15751Abstract Full Text PDF PubMed Google Scholar, 40Graf R. Mattera R. Codina J. Estes M.K. Birnbaumer L. J. Biol. Chem. 1992; 267: 24307-24314Abstract Full Text PDF PubMed Google Scholar, 41Nanoff C. Koppensteiner R. Yang Q. Fuerst E. Ahorn H. Freissmuth M. Mol. Pharmacol. 2006; 69: 397-405Crossref PubMed Scopus (33) Google Scholar, 49Journot L. Pantaloni C. Bockaert J. Audigier Y. J. Biol. Chem. 1991; 266: 9009-9015Abstract Full Text PDF PubMed Google Scholar, 50Denker B.M. Neer E.J. Schmidt C.J. J. Biol. Chem. 1992; 267: 6272-6277Abstract Full Text PDF PubMed Google Scholar), the truncated Gα subunits can form heterotrimers under the experimental conditions used in this study (i.e. in the micromolar range). Only Δ35-Gαi-t showed a complex elution profile with multiple maxima, indicating the presence of larger aggregates. Also, Gβγ had little effect on the retention time, suggesting that Δ35-Gαi-t was unstable or not correctly folded (data not shown).Influence of the N-terminal Region of Gα on the Interaction between the Heterotrimeric G Protein and R*—We applied kinetic light scattering, which is a monitor of R*/G protein interaction (42Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 43Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Crossref PubMed Google Scholar) to study R* binding of nonacylated Gα subunits in the presence of Gβγ (Fig. 3). All full-length Gα subunits investigated, namely rGαt and Gαi-t (Fig. 3A), as well as Gαi (not shown) bound to R* in the presence of Gβγ. Similar binding to R* in the presence of Gβγ was observed for the N-terminal truncated subunits Δ25-rGαt, Δ29-Gαi-t, Δ31-Gαi-t, Δ33-Gαi-t (Fig. 3A), and Δ29-Gαi (not shown). As expected, Gβγ and the apparently misfolded Δ35-Gαi-t did not bind to R* (Fig. 3A, bottom).FIGURE 3Influence of N-terminal deletions of Gα on the interaction between heterotrimeric G proteins and R* monitored by kinetic light scattering. A, light-induced binding of non-fatty acylated Gα subunits (1 μm) in the presence of Gβγ (1 μm) and in the absence of exogenous nucleotides was monitored by kinetic light scattering. Binding is reflected by the increase in the relative intensity of scattered light (ΔI/I) observed after activation of rhodopsin (flash symbol;3 μm rhodopsin in disk membranes; 32% rhodopsin was activated by a green flash, pH 7.4, 23 °C). The Gα subunits investigated are indicated (abbreviated as in the legend to Fig. 1B). B, measurements as in A but in the presence of 50 μm GTP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the presence of Gβγ and excess GTP, light activation of rhodopsin did not give rise to binding signals of Gαi-t, Δ29-Gαi-t, Δ31-Gαi-t, or Δ33-Gαi-t (Fig. 3B). This result indicates that for all four Gα subunits, binding of Gαβγ heterotrimers to R* is followed by rapid GTP uptake and Gαβγ dissociation from both the receptor and the membrane. Furthermore, the lack of any light scattering change suggests that Gαβγ heterotrimers were not bound to the disk membrane in the dark (see Refs. 15Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar and 42Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The same results were obtained with rGαt and Δ25-rGαt as well as with Gαi and Δ29-Gαi (data not shown).As another approach to address receptor-catalyzed G protein activation, GTPγS uptake by Gα subunits in the presence of Gβγ and catalytic amounts of R*-containing disk membranes was monitored by fluorescence spectroscopy (Fig. 4). Consistent with the results from the light scattering assay, the lack of the N-terminal helix in rGαt and Gαi-t had no severe effect on R*-catalyzed nucleotide exchange (Fig. 4). Similar observations were made for Gαi and Δ29-Gαi (data not shown). A gradual decrease in the kinetics was observed as the length of the deletion increased (compare Gαi-t, Δ29-Gαi-t, Δ31-Gαi-t, and Δ33-Gαi-t), which may arise from reduced Gαβγ formation. No GTPγS uptake was observed for Δ35-Gαi-t/Gβγ, again demonstrating the complete functional loss of this Gα mutant.FIGURE 4R*-catalyzed nucleotide exchange of Gα subunits monitored by the fluorescence activa" @default.
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• W2021008077 title "Signal Transfer from GPCRs to G Proteins" @default.
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