A Dominant-Negative Strategy for Studying Roles of G Proteins in Vivo
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6610
ISSN1083-351X
AutoresAnnette Gilchrist, Moritz Bünemann, Anli Li, Marie Thérèse Hosey, Heidi E. Hamm,
Tópico(s)Cellular transport and secretion
ResumoG proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the α subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein α subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Gαi, Gαq, or Gαs. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Gαi carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Gαi minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Gα carboxyl peptide in random order, pcDNA-GαiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Gαi minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Gαq or Gαs had no effect on M2 muscarinic receptor stimulation of the K+channel. G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the α subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein α subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Gαi, Gαq, or Gαs. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Gαi carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Gαi minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Gα carboxyl peptide in random order, pcDNA-GαiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Gαi minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Gαq or Gαs had no effect on M2 muscarinic receptor stimulation of the K+channel. Many biologically active molecules transduce their signals through heptahelical receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). 1The abbreviations used are: G proteins, guanine nucleotide-binding proteins; GPCR, G protein-coupled receptor; IP, inositol phosphate; HEK, human embryonic kidney; mAChR, muscarinic receptor; ACh, acetylcholine; IKACh, inwardly rectifying K+ channel; GIRK, G protein-regulated inwardly rectifying K+ channel; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s).1The abbreviations used are: G proteins, guanine nucleotide-binding proteins; GPCR, G protein-coupled receptor; IP, inositol phosphate; HEK, human embryonic kidney; mAChR, muscarinic receptor; ACh, acetylcholine; IKACh, inwardly rectifying K+ channel; GIRK, G protein-regulated inwardly rectifying K+ channel; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s). G proteins play important roles in determining the specificity and temporal characteristics of a variety of cellular responses. Upon activation, G protein-coupled receptors (GPCRs) interact with their cognate heterotrimeric G protein, inducing GDP release with subsequent GTP binding to the α subunit. The exchange of GDP for GTP leads to dissociation of the Gβγ dimer from the Gα subunit, and both initiate unique intracellular signaling responses (for review, see Refs. 1Hamm H.E. Gilchrist A. Curr. Opin. Cell Biol. 1996; 8: 189-196Crossref PubMed Scopus (203) Google Scholar and 2Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar). Molecular cloning has resulted in the identification of 18 distinct Gα subunits that are commonly divided into four families based on their sequence similarity: Gi, Gs, Gq, and G12. Similarly, multiple Gβ (5Slepak V.Z. Quick M.W. Aragay A.M. Davidson N. Lester H.A. Simon M.I. J. Biol. Chem. 1993; 268: 21889-21894Abstract Full Text PDF PubMed Google Scholar) and Gγ (11Higashijima T. Ferguson K.M. Sternweis P.C. Smigel M.D. Gilman A.G. J. Biol. Chem. 1987; 262: 762-766Abstract Full Text PDF PubMed Google Scholar) subunits have been identified.In all G proteins studied GTP is bound as a complex with Mg2+, and the GTP- and Mg2+-binding sites are tightly coupled. Dominant-negative constructs of the α subunit of G proteins have been made in which mutations are made in residues that contact the magnesium ion. Although this approach was quite successful with p21ras and other small G proteins (3John J. Rensland H. Schlichting I. Vetter I. Borasio G.D. Goody R.S. Wittinghofer A. J. Biol. Chem. 1993; 268: 923-929Abstract Full Text PDF PubMed Google Scholar, 4Quilliam L.A. Kato K. Rabun K.M. Hisaka M.M. Huff S.Y. Campbell-Burk S. Der C.I. Mol. Cell. Biol. 1994; 14: 1113-1121Crossref PubMed Scopus (80) Google Scholar), dominant-negative Gαi, Gαo, Gαq, and Gα11 have been less effective (5Slepak V.Z. Quick M.W. Aragay A.M. Davidson N. Lester H.A. Simon M.I. J. Biol. Chem. 1993; 268: 21889-21894Abstract Full Text PDF PubMed Google Scholar, 6Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 7Osawa S. Johnson G.L. J. Biol. Chem. 1991; 266: 4673-4676Abstract Full Text PDF PubMed Google Scholar, 8Hermouet S. Merendino J.J. Gutkind J.S. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10455-10459Crossref PubMed Scopus (104) Google Scholar, 9Carrel F. Dharmawardhane S. Clark A.M. Powell-Coffman J.A. Firtel R.A. Mol. Biol. Cell. 1994; 5: 7-16Crossref PubMed Scopus (9) Google Scholar, 10Winitz S. Gupta S.K. Qian N.-X. Heasley L.E. Nemenoff R.A. Johnson G.L. J. Biol. Chem. 1994; 269: 1889-1895Abstract Full Text PDF PubMed Google Scholar). This is probably because of the degree to which Mg2+ is necessary to support GDP binding. p21ras forms a tight and nearly irreversible GDP·Mg2+ complex, whereas Gα subunits bind Mg2+ in the GDP·Mg2+ complex with lower affinity than in the GTP·Mg2+ complex (11Higashijima T. Ferguson K.M. Sternweis P.C. Smigel M.D. Gilman A.G. J. Biol. Chem. 1987; 262: 762-766Abstract Full Text PDF PubMed Google Scholar, 12Higashijima T. Ferguson K.M. Smigel M.D. Gilman A.G. J. Biol. Chem. 1987; 262: 757-761Abstract Full Text PDF PubMed Google Scholar, 13Lee E. Taussig R. Gilman A.G. J. Biol. Chem. 1992; 267: 1212-1218Abstract Full Text PDF PubMed Google Scholar, 14Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (879) Google Scholar).Specific determinants of receptor-G protein interaction have been under investigation for many years. It is thought that there are multiple sites of contact between the activated receptor and the G protein. Studies using ADP-ribosylation by pertussis toxin, site-directed mutagenesis, peptide-specific antibodies, and chimeric proteins indicate that the carboxyl terminus of the Gα subunit is not only an essential region for receptor contact, but is also important for determining G protein receptor specificity (reviewed in Refs. 1Hamm H.E. Gilchrist A. Curr. Opin. Cell Biol. 1996; 8: 189-196Crossref PubMed Scopus (203) Google Scholar and15Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar). The crystal structures of various Gα subunits show that the last 4–7 amino acids of Gα were not observed (16Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (530) Google Scholar, 17Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (522) Google Scholar, 18Lambright 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, 19Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar, 20Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1005) Google Scholar, 21Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 269: 1405-1412Crossref Scopus (747) Google Scholar, 22Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (267) Google Scholar) indicating that the region is conformationally flexible in the absence of other interactions.In vitro assays, as well as microinjection studies of intact cells, indicate Gα carboxyl-terminal peptides can competitively block G protein-coupled downstream events (23Hamm H.E. Deretic D. Hofman K. Schleicher A. Kohl B. J. Biol. Chem. 1987; 262: 10831-10838Abstract Full Text PDF PubMed Google Scholar, 24Takano K. Yasufuku-Takano J. Kozasa T. Nakajima S. Nakajima Y. J. Physiol. 1997; 502: 559-567Crossref PubMed Scopus (40) Google Scholar, 25Aragay A.M. Collins L.R. Post G.R. Watson A.J. Feranisco J.P. Brown J.H. Simon M.I. J. Biol. Chem. 1995; 270: 20073-20077Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 26Jones S. Brown D.A. Milligan G. Willer E. Buckley N.J. Caulfield M.P. Neuron. 1995; 14: 399-405Abstract Full Text PDF PubMed Scopus (122) Google Scholar). A carboxyl-terminal peptide from Gαt not only binds, but will also directly stabilize photoactivated rhodopsin (27Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hoffmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (390) Google Scholar, 28Osawa S. Weiss E.R. J. Biol. Chem. 1995; 270: 31052-31058Crossref PubMed Scopus (65) Google Scholar). Using a combinatorial peptide library Martin et al. (29Martin E.L. Rens-Domiano S. Schatz P.J. Hamm H.E. J. Biol. Chem. 1996; 271: 361-366Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) have shown that specific residues within the carboxyl terminus of Gαt are critical for high affinity binding of the Gαt peptide to rhodopsin. Similarly, a carboxyl-terminal peptide from Gαs (384–394), but not corresponding peptides from Gαi1/2, inhibits the ability of β2-adrenergic receptors to activate Gαs and adenylyl cyclase (30Rasenick M.M. Watanabe M. Lazarevic M.B. Hatta S. Hamm H.E. J. Biol. Chem. 1994; 269: 21519-21525Abstract Full Text PDF PubMed Google Scholar). In addition, a carboxyl-terminal undecapeptide from Gαi1/2 can bind the adenosine A1 receptor, whereas the corresponding peptide from Gαt, which differs by only 1 amino acid residue does not (31Gilchrist A. Mazzoni M. Dineen B. Dice A. Linden J. Proctor W.R. Lupica C.R. Dunwiddie T. Hamm H.E. J. Biol. Chem. 1998; 273: 14912-14919Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Thus, the carboxyl terminus of G protein α subunits is critical in both mediating receptor-G protein interactions and in receptor selectivity (31Gilchrist A. Mazzoni M. Dineen B. Dice A. Linden J. Proctor W.R. Lupica C.R. Dunwiddie T. Hamm H.E. J. Biol. Chem. 1998; 273: 14912-14919Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 32Blahos J.N. Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 33Kostenis E. Conklin B.R. Wess J. Biochemistry. 1997; 36: 1487-1495Crossref PubMed Scopus (96) Google Scholar, 34Liu J. Conklin B.R. Blin N. Yun J. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11642-11646Crossref PubMed Scopus (195) Google Scholar, 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)."Minigene" plasmid vectors are constructs designed to express relatively short polypeptide sequences following their transfection into mammalian cells. Minigenes have been used by investigators to look at a variety of responses related to G proteins including (i) binding of pleckstrin homology (PH) domains to Gβγ (36Luttrell L.M. Hawes B.E. Touhara K. van Biesen T. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), (ii) inhibiting GPCRs by expressing the carboxyl terminus of β2 adrenergic receptor kinase (37Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar, 38Dickenson J.M. Hill S.J. Eur. J. Pharmacol. 1998; 355: 85-93Crossref PubMed Scopus (66) Google Scholar, 39Fedorov Y.V. Jones N.C. Olwin B.B. Mol. Cell. Biol. 1998; 18: 5780-5787Crossref PubMed Scopus (49) Google Scholar), and (iii) identifying intracellular domains of GPCRs critical for G protein coupling (40Hawes B.E. Luttrell L.M. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 15776-15785Abstract Full Text PDF PubMed Google Scholar, 41Luttrell L.M. Ostrowski J. Cotecchia S. Kendall H. Lefkowitz R.J. Science. 1993; 259: 1453-1457Crossref PubMed Scopus (89) Google Scholar, 42Carlson S.A. Chatterjee T.K. Fisher R.A. J. Biol. Chem. 1996; 271: 23146-23153Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 43Thompson J.B. Wade S.M. Harrison J.K. Salafranca M.N. Neubig R.R. J. Pharmacol. Exp. Ther. 1998; 285: 216-222PubMed Google Scholar, 44Ulloa-Aguirre A. Stanislaus D. Arora V. Vaananen J. Brothers S. Janovick J.A. Conn P.M. Endocrinology. 1998; 139: 2472-2478Crossref PubMed Google Scholar). Experiments using minigenes that express the last 55 amino acids of Gαq to target the receptor-Gq interface to achieve class-specific inhibition were recently published by Akhter et al. (45Akhter S.A. Luttrell L.M. Rockman H.A. Iaccarino G. Lefkowitz R.J. Koch W.J. Science. 1998; 280: 574-577Crossref PubMed Scopus (393) Google Scholar). Transient transfection of COS-7 cells with α1B-adrenergic receptors or M1 muscarinic receptors and the Gαqcarboxyl-terminal minigene (residues 305–359) inhibits agonist stimulated inositol phosphate (IP) production, whereas co-expression with the Gαq amino terminus (residues 1–54) has no effect. Inhibition by Gαq (305–359) was apparently specific for Gq-coupled receptors because neither α2A-adrenergic receptor-mediated IP production (Gi-coupled), nor dopamine D1Areceptor-mediated cAMP production (Gs-coupled) were inhibited. In addition, transgenic mice made by targeting the Gαq carboxyl-terminal minigene to the myocardium resulted in a marked inhibition of α1B-adrenergic receptor-mediated IP production and blockade of cardiac hypertrophy.In this paper, we study the effects of several carboxyl termini Gα peptides using a minigene approach. To test whether minigene constructs encoding the carboxyl-terminal 11 amino acid residues from Gα subunits could effectively inhibit G protein-coupled receptor-mediated cellular responses, we chose a system in which 1) the importance of the carboxyl terminus and 2) the downstream effector system had been well established. Numerous studies (46Lai J. Waite S.L. Bloom J.W. Yamamura H.I. Roeske W.R. J. Pharmacol. Exp. Ther. 1991; 258: 938-944PubMed Google Scholar, 47Thomas E.A. Ehlert F.J. J. Pharmacol. Exp. Ther. 1994; 271: 1042-1050PubMed Google Scholar, 48Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar, 49Dell'Acqua M.L. Carroll R.C. Peralta E.G. J. Biol. Chem. 1993; 268: 5676-5685Abstract Full Text PDF PubMed Google Scholar) have shown that the M2 muscarinic receptor (mAChR) couples exclusively to the Gi/Go family. The M2 mAChR can efficiently couple to mutant Gαq in which the last 5 amino acids of Gαq are substituted with the corresponding residues from Gαi or Gαo (34Liu J. Conklin B.R. Blin N. Yun J. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11642-11646Crossref PubMed Scopus (195) Google Scholar), suggesting that this receptor contains domains that are specifically recognized by the carboxyl terminus of Gαi/o subunits. The effector system that we selected was the M2 mAChR-activated inwardly rectifying K+ channel (IKACh). In cardiac cells, the IKACh channel is formed as a heterotetramer of G protein-regulated inwardly rectifying K+ channels (GIRK), with two GIRK1 and two GIRK4 subunits (50Corey S. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 5271-5278Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 51Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (751) Google Scholar). This channel is activated upon stimulation of M2 mAChR in a manner that is completely pertussis toxin-sensitive and is the prototype for a direct Gβγ-activated channel (52Krapivinsky G. Krapivinsky L. Wickman K. Clapham D.E. J. Biol. Chem. 1995; 270: 29059-29062Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 53Krapivinsky G. Kennedy M.E. Nemec J. Medina I. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 16946-16952Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 54Sowell M.O. Ye C. Ricupero D.A. Hansen S. Quinn S. Vassilev P. Mortensen R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7921-7926Crossref PubMed Scopus (42) Google Scholar). Our experiments indicate that the Gαi carboxyl terminus minigene construct can completely block M2 mAChR-mediated K+ channel activation. The inhibition appears specific as constructs producing Gαs, Gαq, or a scrambled Gαicarboxyl-terminal peptide had no effect.RESULTS AND DISCUSSIONDominant-negative constructs of the α subunit of G proteins have been made in which mutations are made in regions that contact the magnesium ion. For the α subunit of G proteins, this includes mutations of the Gly residue within the invariant sequence (G203T,G204A), as well as mutations of a Ser residue in the effector loop, switch I region (S47C) in either Gαo or Gαi (5Slepak V.Z. Quick M.W. Aragay A.M. Davidson N. Lester H.A. Simon M.I. J. Biol. Chem. 1993; 268: 21889-21894Abstract Full Text PDF PubMed Google Scholar, 6Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, neither of these mutations has resulted in effective dominant-negatives probably because the GDP complexes of Gαi, Gαo, and Gαs have low affinity for Mg2+. Thus, we looked to other regions on G protein α subunits that could serve to block receptor-G protein interactions, and consequently serve as dominant-negatives. A variety of studies have implicated the carboxyl terminus of G protein α subunits in mediating receptor-G protein interaction and selectivity (for review, see Refs. 14Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (879) Google Scholar and 15Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar). We have shown that carboxyl termini from G protein α subunits are important sites of receptor binding, and peptides corresponding to the carboxyl terminus can be used as competitive inhibitors of receptor-G protein interactions (27Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hoffmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (390) Google Scholar, 29Martin E.L. Rens-Domiano S. Schatz P.J. Hamm H.E. J. Biol. Chem. 1996; 271: 361-366Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 30Rasenick M.M. Watanabe M. Lazarevic M.B. Hatta S. Hamm H.E. J. Biol. Chem. 1994; 269: 21519-21525Abstract Full Text PDF PubMed Google Scholar). This interaction is quite specific as we found that a difference in 1 amino acid can annul the ability of the Gαi1/2 peptide to bind the A1 adenosine receptor-G protein interface (31Gilchrist A. Mazzoni M. Dineen B. Dice A. Linden J. Proctor W.R. Lupica C.R. Dunwiddie T. Hamm H.E. J. Biol. Chem. 1998; 273: 14912-14919Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).To determine whether we could selectively antagonize G protein signal transduction events in vivo by expressing peptides that block the receptor-G protein interface, we generated minigene plasmid constructs that encode carboxyl-terminal peptide sequences from Gαi1/2, Gαq, or Gαs (TableI). As a control, we also made a minigene that expressed the carboxyl terminus of Gαi1/2 in random order (GαiR, Table I). The minigene insert DNA were made by synthesizing short complimentary oligonucleotides corresponding to the peptide sequences from the carboxyl terminus of each Gα withBamHI and HindIII restriction sites at the 5′ and 3′ ends, respectively. Complementary oligonucleotides were annealed and ligated into the mammalian expression vector pcDNA 3.1(−). The DNA was cut with NcoI, and separated on a 1.5% agarose gel to determine whether the insert was present. As shown in Fig.1, when insert is present there is a newNcoI site resulting in a shift in the band pattern, such that the digest pattern goes from three bands (3345, 1352, and 735 bp) to four bands (3345, 1011, 735, and 380 bp).Table ICarboxyl termini sequencesGαl1/2I K N N L K D C G L FGαqL Q L N L K E Y N A VGαsQ R M H L R Q Y E L LGαiRN G I K C L F N D K LAlignment of the last 11 amino acid residues from human Gαi1/2, Gαq, and Gαs subunits. Also shown is the peptide sequence of GαiR, the Gαi1/2sequence in random order, used to construct the control minigene. Open table in a new tab As our minigene approach depends on competitive inhibition, a key element for success is the expression of adequate amounts of peptides to block intracellular signaling pathways. To confirm the presence of the minigene constructs in transfected cells, total RNA was isolated 48 h posttransfection, cDNA made with RT-PCR, and PCR analysis was performed using the cDNA as template with primers specific for the Gα carboxyl-terminal peptide insert. Separation of the PCR products on 1.5% agarose gels (Fig.2 A) indicates the presence of the Gα carboxyl terminus peptide minigene RNA by a single 434-bp band. Control experiments were done using a T7 forward primer with the vector reverse primer to verify the presence of the pcDNA3.1 vector, and G3DPH primers (CLONTECH) to approximate the amount of total RNA (data not shown).To verify that the peptide was being produced in the transfected cells, 48 h posttransfection, cells were lysed and homogenized. Cytosolic extracts were analyzed by high pressure liquid chromatography, and peaks (Fig. 2 B) were analyzed by ion mass spray analysis. The mass spectrometer analysis for peak 1 from the pcDNA-Gαi transfected cells, and peak 1 from cells transfected with a vector expressing the carboxyl terminus in random order (pcDNA-GαiR) indicate that a 1450 molecular weight peptide was found in both cytosolic extracts. This is the expected molecular weight for both 13 amino acid peptide sequences. The fact that they were the major peptides found in the cytosol from cells transiently transfected with the pcDNA-Gαi or pcDNA-GαiR vectors strongly suggests that the vectors are producing the appropriate peptide sequences. Therefore, analysis of the transiently transfected HEK 293 cells indicates (1Hamm H.E. Gilchrist A. Curr. Opin. Cell Biol. 1996; 8: 189-196Crossref PubMed Scopus (203) Google Scholar) minigene vectors are present, and (2Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar) the corresponding peptides are being expressed.We examined whether the presence of the Gαicarboxyl-terminal peptide minigene would result in a significant inhibition of a downstream functional response following agonist stimulation of the transiently transfected cells. G protein-regulated inwardly rectifying K+ channels modulate electrical activity in many excitable cells (for review, see Refs. 60Jan L.Y. Jan Y.N. Curr. Opin. Cell Biol. 1997; 9: 155-160Crossref PubMed Scopus (34) Google Scholar, 61Breitwieser G. J. Membr. Biol. 1996; 152: 1-11Crossref PubMed Scopus (40) Google Scholar, 62Wickman K.B. Clapham D.E. Curr. Opin. Neurobiol. 1995; 5: 278-285Crossref PubMed Scopus (70) Google Scholar). Because the channel opens as a consequence of a direct interaction with Gβγ, whole cell patch clamp recording of inwardly rectifying K+ currents can be used as a readout of G protein activity in single intact cells. Thus, we tested whether the Gα carboxyl-terminal peptide minigenes could inhibit M2 mAChR activation of inwardly rectifying K+ currents. Superfusion of HEK 293 cells transiently transfected with GIRK1/GIRK4 and either pcDNA-Gαi or pcDNA-GαiR DNA with 1 μm ACh revealed that cells transfected with pcDNA-Gαi DNA have a dramatically impaired response to the M2 mAchR agonist (Fig.3). Fig. 3, A and Bshows representative recordings of whole cell membrane currents at −90 mV. Superfusion of the cells with ACh activates inward currents in cells transfected with pcDNA-GαiR (Fig.3 A) but not in cells transfected with pcDNA-Gαi (Fig. 3 B). The inwardly rectifying IV-curve for the ACh-induced current from the experiment shown in Fig. 3 A is illustrated in Fig. 3 D. The strong inwardly rectifying properties of this current is characteristic of IKACh channels. Summarized data for the maximum amplitude of ACh-evoked currents are shown for three different transfection conditions as indicated by the black bars. The maximum current evoked by ACh was 3.7 ± 1.5 pA/pF (n = 14) in cells transfected with the pcDNA-Gαi compared with 24.1 ± 8.8 pA/pF (n = 11) in cells transfected with pcDNA-GαiR. As a control we transfected cells with empty vector (pcDNA3.1). The ACh responses in these cells (16.5 ± 7.7 pA/pF (n = 5) was not significantly different from responses measured in cells transfected with pcDNA-GαiR (Fig. 3 C). Basal levels for all three conditions were equivalent (pcDNA 3.2 ± 1.8 pA/pF (n = 5); Gαi 6.1 ± 0.9 pA/pF (n = 14); GαiR 5.6 ± 2.0 pA/pF (n = 10)). To exclude experiments in which we recorded currents from cells that may not have expressed the functional channel, only those cells that exhibited a basal nonagonist-dependent Ba2+ (200 μm) sensitive inwardly rectifying current were used for analysis. Thus, it appears that the Gαi minigene construct completely blocks the agonist-mediated M2 mAChR GIRK1/4 response, whereas the control minigene constructs (empty vector, pcDNA3.1, and the Gαi1/2 carboxyl peptide in random order, pcDNA-GαiR) had no effect on the agonist-mediated M2 mAChR GIRK1/4 response.Figure 3Minigenes encoding carboxyl-terminal Gαi peptides inhibit M2mAChR activated IKACh. HEK 293 cells stably expressing the M2 mAChR were transiently transfected with DNA from GIRK1/4 and pcDNA3.1, pcDNA-Gαi, or pcDNA-GαiR. A, a representative example of the activation of inwardly rectifying K+ currents upon superfusion of 1 μm ACh in a HEK 293 cell transiently transfected with GIRK1, GIRK4, and pcDNA-GαiR DNA.B, a representative example of the activation of inwardly rectifying K+ currents upon superfusion of 1 μm ACh in a HEK 293 cell transiently transfected with GIRK1, GIRK4, and pcDNA-Gαi DNA. C, the maximum current evoked by Ach (black bars) was 3.7 ± 1.5 pA/pF (n = 14) in cells transfected with the pcDNA-Gαi compared with 16.5 ± 7.7 pA/pF (n = 5) in cells transfected with empty vector (pcDNA3.1) or 24.1 ± 8.8 pA/pF (n = 11) in cells transfected with pcDNA-GαiR. The white bars represent the basal Ba2+-sensitive currents.D, the current voltage relation of the ACh-induced current shows the characteristic inward rectification and a reversal potential near the potassium equilibrium potential typical for IKACh.View Large Image Figure ViewerDownload (PPT)The cardiac IKACh channel is activated upon stimulation of M2 mAChR via G proteins of the Gi family. The carboxyl-terminal region of Gα has also been shown to be critical in determining the specificity of GPCR-G protein interactions (34Liu J. Conklin B.R. Blin N. Yun J. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11642-11646Crossref PubMed Scopus (195) Google Scholar, 63Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (601) Google Scholar). Substitution of 3–5 carboxyl-terminal amino acids from Gαq with corresponding residues from Gαiallowed receptors that signal exclusively through Gαisubunits to activate the chimeri
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