The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

Abstract

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways. G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways. G protein–coupled receptors (GPCRs), also called 7-transmembrane receptors, are the largest group of membrane proteins that regulate a multitude of physiological processes (1Pierce K.L. Premont R.T. Lefkowitz R.J. Seven-transmembrane receptors.Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2164) Google Scholar, 2Rockman H.A. Koch W.J. Lefkowitz R.J. Seven-transmembrane-spanning receptors and heart function.Nature. 2002; 415: 206-212Crossref PubMed Scopus (797) Google Scholar). Approximately 35% of all Food and Drug Administration–approved drugs target GPCRs (3Sriram K. Insel P.A. G protein-coupled receptors as targets for approved drugs: How many targets and how many drugs?.Mol. Pharmacol. 2018; 93: 251-258Crossref PubMed Scopus (662) Google Scholar). In response to various external stimuli, such as light, hormones, and neurotransmitters, GPCRs initiate diverse cellular signaling processes through G protein–dependent and β-arrestin (βarr)-dependent pathways (4Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (519) Google Scholar, 5DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Beta-arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1194) Google Scholar). In the classical G protein–dependent pathway, agonist-bound GPCRs activate heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to cellular response through a range of second messengers and other effectors (6Oldham W.M. Hamm H.E. Heterotrimeric G protein activation by G-protein-coupled receptors.Nat. Rev. Mol. Cell Biol. 2008; 9: 60-71Crossref PubMed Scopus (856) Google Scholar, 7Gilman A.G. 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Lefkowitz R.J. beta-Arrestin: a protein that regulates beta-adrenergic receptor function.Science. 1990; 248: 1547-1550Crossref PubMed Scopus (931) Google Scholar). Furthermore, βarrs coupling to the activated GPCR initiates alternative signaling pathways independent of or in concert with G proteins (5DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Beta-arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1194) Google Scholar, 11Thomsen A.R.B. Plouffe B. Cahill 3rd, T.J. Shukla A.K. Tarrasch J.T. Dosey A.M. Kahsai A.W. Strachan R.T. Pani B. Mahoney J.P. Huang L. Breton B. Heydenreich F.M. Sunahara R.K. Skiniotis G. et al.GPCR-G protein-beta-arrestin super-complex mediates sustained G protein signaling.Cell. 2016; 166: 907-919Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). βarrs thus serve as adaptors and scaffold proteins that link GPCR to numerous signaling molecules, such as components of mitogen-activated protein kinase (MAPK) cascades and nonreceptor tyrosine kinase Src, and regulate various cellular functions, such as chemotaxis and apoptosis (12Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.Science. 1999; 283: 655-661Crossref PubMed Scopus (1287) Google Scholar, 13Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar, 15Peterson Y.K. Luttrell L.M. The diverse roles of arrestin scaffolds in G protein-coupled receptor signaling.Pharmacol. Rev. 2017; 69: 256-297Crossref PubMed Scopus (287) Google Scholar). MAPK cascades play a critical role in transmitting environmental signals into cellular responses (16Davis R.J. The mitogen-activated protein kinase signal transduction pathway.J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). A typical MAPK cascade consists of three types of kinases: an MAP kinase kinase kinase, an MAP kinase kinase, and an MAPK (17Plotnikov A. Zehorai E. Procaccia S. Seger R. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation.Biochim. Biophys. Acta. 2011; 1813: 1619-1633Crossref PubMed Scopus (668) Google Scholar). In the typical Raf–MAPK extracellular signal–regulated kinase (MEK)–extracellular signal-regulated kinase (ERK) module, activated C-Raf (MAP kinase kinase kinase) phosphorylates downstream MEK1 (also known as MAP kinase kinase), which then phosphorylates the ERK (MAPK). ERK then phosphorylates numerous effector proteins and regulates diverse physiological processes, such as cell growth, proliferation, and differentiation (18Seger R. Krebs E.G. The MAPK signaling cascade.FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3254) Google Scholar). C-Raf is the most upstream kinase in the cascade, and its activation is crucial for MAPK signaling. C-Raf consists of three conserved regions (CRs) (Fig. 1A). CR1, on the amino terminus, contains a Ras-binding domain (RBD) and a cysteine-rich domain (CRD); CR2 corresponds to a region rich in serine/threonine residues, whereas CR3 includes the catalytic domain on the carboxyl terminus (19Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (426) Google Scholar). The activation of C-Raf is a complex process that involves Ras binding, regulatory phosphorylation, and protein–lipid interactions (19Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (426) Google Scholar, 20Terrell E.M. Morrison D.K. Ras-mediated activation of the Raf family kinases.Cold Spring Harb. Perspect. Med. 2019; 9a033746Crossref PubMed Scopus (97) Google Scholar). GPCR–βarr complexes interact with C-Raf to initiate MAPK cascade signaling (13Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar). Both the N and C domains of βarrs have been known to interact with C-Raf (21Song X. Coffa S. Fu H. Gurevich V.V. How does arrestin assemble MAPKs into a signaling complex?.J. Biol. Chem. 2009; 284: 685-695Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). However, the βarr-binding site in C-Raf has not been determined. βarrs have been demonstrated to act like typical scaffold proteins that bring together the components of MAPK cascade to facilitate signal transduction (13Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (712) Google Scholar, 14DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar). Surprisingly, recent studies showed that in addition to scaffolding functions, GPCR–βarr complexes may serve as allosteric modulators for downstream signaling partners such as kinase Src (22Pakharukova N. Masoudi A. Pani B. Staus D.P. Lefkowitz R.J. Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.J. Biol. Chem. 2020; 295: 16773-16784Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Yang F. Xiao P. Qu C.X. Liu Q. Wang L.Y. Liu Z.X. He Q.T. Liu C. Xu J.Y. Li R.R. Li M.J. Li Q. Guo X.Z. Yang Z.Y. He D.F. et al.Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.Nat. Chem. Biol. 2018; 14: 876-886Crossref PubMed Scopus (44) Google Scholar). However, it is unclear whether βarrs can also allosterically activate C-Raf. Here, we show that βarr1, activated via either phosphorylated GPCR or the GPCR surrogate V2Rpp (a vasopressin receptor 2 phosphorylated C-terminal peptide with eight phosphates), binds C-Raf and allosterically activates it. Therefore, GPCR–βarr complexes not only function just as typical scaffold proteins for the C-Raf/MEK/ERK module but also play an active role in regulating MAPK signaling. To identify the βarr1-binding elements within C-Raf, we performed glutathione-S-transferase (GST) pull-down assays using GST-C-Raf fusion proteins and βarr1 (Fig. 1A). βarr1 with the GST-C-Raf amino terminus, and βarr1 was for GST-C-Raf carboxyl terminus (Fig. show that does not interact with βarr1 βarr1 and C-Raf amino terminus interact Both and active βarr1 bind C-Raf amino the binding was in the presence of V2Rpp and the The amino terminus of C-Raf the and To the roles of domains in the and contains the and as proteins to the with βarr1, binds βarr1 (Fig. that the domain of C-Raf directly interacts with In the presence of the domain for and of the thus to the binding of Interestingly, H-Ras, a small GTPase known to activate C-Raf, also interacts with the same domains of C-Raf S. S. Raf domains mediate interaction with Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). To whether binding for H-Ras and βarr1 have we an H-Ras with (a analog of can with βarr1 to bind an excess of activated H-Ras, the interaction between and βarr1 was reduced (Fig. with binding of βarr1 and H-Ras to the same region of C-Raf. Therefore, these data suggest that βarr1 binds to the amino terminus of C-Raf as GPCR–βarr complexes have been demonstrated to allosterically activate signaling partners such as the tyrosine kinase Src (22Pakharukova N. Masoudi A. Pani B. Staus D.P. Lefkowitz R.J. Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.J. Biol. Chem. 2020; 295: 16773-16784Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Yang F. Xiao P. Qu C.X. Liu Q. Wang L.Y. Liu Z.X. He Q.T. Liu C. Xu J.Y. Li R.R. Li M.J. Li Q. Guo X.Z. Yang Z.Y. He D.F. et al.Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.Nat. Chem. Biol. 2018; 14: 876-886Crossref PubMed Scopus (44) Google Scholar). To whether GPCR–βarr complexes allosterically regulate C-Raf we an to the C-Raf in (Fig. To the scaffolding function of βarr1, we the of protein of the physiological by C-Raf. We a receptor with a phosphorylated C-tail of the vasopressin 2 receptor that binding to was in membrane scaffold protein as a and it a complex on the D.P. H. M.J. L.M. Lefkowitz R.J. Skiniotis G. Structure of the complex in a 2020; PubMed Scopus Google Scholar). GPCR–βarr complexes such as the are by a synthetic The C-Raf catalytic in the presence of the as shown in (Fig. and as (Fig. of C-Raf The and or to the C-Raf To whether the complex activates C-Raf, we a in kinase using to the of by C-Raf in the or the presence of We found that the complex the C-Raf in with (Fig. Therefore, reveal that the complex allosterically activates kinase C-Raf. we an excess of the C-Raf amino terminus to its to the allosteric activation of C-Raf by The allosteric activation of C-Raf is by excess C-Raf amino terminus (Fig. reveal that the complex allosterically activates C-Raf by with its amino terminus. data also demonstrate that βarr1 binds to the amino terminus of C-Raf. studies that GPCR–βarr complexes in the βarrs bind to the phosphorylated C terminus and the of in the βarrs bind only to the phosphorylated GPCR C terminus A.W. Pani B. Lefkowitz R.J. GPCR activation of 2018; PubMed Scopus Google Scholar). studies show that the of βarr1 interacts with the and as a membrane (Fig. D.P. H. M.J. L.M. Lefkowitz R.J. Skiniotis G. Structure of the complex in a 2020; PubMed Scopus Google Scholar, B. J. M.E. of arrestin function as a membrane 2017; PubMed Scopus Google Scholar). the βarr1 which is required for interactions with the receptor core, we a complex in the 3rd, T.J. Tarrasch J.T. Plouffe B. Yang F. Huang L.Y. Kahsai A.W. S. Shukla A.K. B. et of GPCR-beta-arrestin complexes mediate and Natl. Acad. Sci. U. S. A. 2017; PubMed Scopus Google Scholar). in the of βarr1, we a complex that interactions with the (Fig. Both and complexes showed to C-Raf with the complex (Fig. suggest that the interactions between the GPCR and βarr1 between the of βarr1 and the are necessary for the allosteric activation of C-Raf by suggest that the binding of the phosphorylated GPCR C terminus to βarr1 a critical role in the allosteric activation of C-Raf. To V2Rpp was to activate βarr1 in vitro. We found that the active βarr1 allosterically activate C-Raf, whereas βarr1 to also show that V2Rpp was sufficient to activate C-Raf, whereas not have a to (Fig. demonstrate that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for the allosteric activation of C-Raf. H-Ras a critical role in the activation process of C-Raf (19Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (426) Google Scholar, 20Terrell E.M. Morrison D.K. Ras-mediated activation of the Raf family kinases.Cold Spring Harb. Perspect. Med. 2019; 9a033746Crossref PubMed Scopus (97) Google Scholar). To the allosteric activation between GPCR–βarr complexes and activated H-Ras on C-Raf, we the C-Raf in the presence of the complex or The and of C-Raf show that and H-Ras the catalytic of C-Raf (Fig. The activation of H-Ras is but that of the C-Raf is but not the and H-Ras are together (Fig. In the analog was the process to C-Raf. show that the complex or H-Ras allosterically activate C-Raf in a Taken together, demonstrate that in addition to the canonical scaffolding the GPCR–βarr complexes serve as direct allosteric of C-Raf. βarrs have been demonstrated to interact with the C-Raf/MEK/ERK cascade coupling to GPCRs and mediate activation of pathway either independent of or on G proteins A.R.B. Plouffe B. Cahill 3rd, T.J. Shukla A.K. Tarrasch J.T. Dosey A.M. Kahsai A.W. Strachan R.T. Pani B. Mahoney J.P. Huang L. Breton B. Heydenreich F.M. Sunahara R.K. Skiniotis G. et al.GPCR-G protein-beta-arrestin super-complex mediates sustained G protein signaling.Cell. 2016; 166: 907-919Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 15Peterson Y.K. Luttrell L.M. The diverse roles of arrestin scaffolds in G protein-coupled receptor signaling.Pharmacol. Rev. 2017; 69: 256-297Crossref PubMed Scopus (287) Google Scholar). have that C-Raf interacts with the N and C domains of βarr1 (21Song X. Coffa S. Fu H. Gurevich V.V. How does arrestin assemble MAPKs into a signaling complex?.J. Biol. Chem. 2009; 284: 685-695Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, S. M. Gurevich V.V. A in activation by 2011; PubMed Scopus Google Scholar, Q.T. Xiao P. Huang S.M. Lin J.Y. Yang F. R.J. Xiao Wang J. C. J.P. et studies of interactions between the receptor and PubMed Scopus Google Scholar). A of module complexes by that βarr1 binds to the of C-Raf F. E. P. J. A. the of a G protein-coupled module 2015; PubMed Scopus Google Scholar). In the using pull-down we demonstrated that the domain of C-Raf interacts with In we found that activated H-Ras with βarr1 for binding to C-Raf. activated H-Ras is thought to bring C-Raf to the membrane for Ras in Raf activation is by targeting Raf to the PubMed Scopus Google GPCR–βarr complexes play a role as an of the to the cell membrane the activation process of C-Raf. βarrs have been adaptors and scaffold proteins that link the receptors to signaling partners and bring various signaling components into to facilitate the signal transduction process Y.K. Luttrell L.M. The diverse roles of arrestin scaffolds in G protein-coupled receptor signaling.Pharmacol. Rev. 2017; 69: 256-297Crossref PubMed Scopus (287) Google Scholar). However, recent studies have shown that βarr1 can play a active role in signaling by allosterically its binding Src kinase (22Pakharukova N. Masoudi A. Pani B. Staus D.P. Lefkowitz R.J. Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.J. Biol. Chem. 2020; 295: 16773-16784Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Yang F. Xiao P. Qu C.X. Liu Q. Wang L.Y. Liu Z.X. He Q.T. Liu C. Xu J.Y. Li R.R. Li M.J. Li Q. Guo X.Z. Yang Z.Y. He D.F. et al.Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.Nat. Chem. Biol. 2018; 14: 876-886Crossref PubMed Scopus (44) Google Scholar). However, for the MAPK signaling pathway, the of how C-Raf is to initiate the signaling cascade that βarr-dependent MAPK signaling a process K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (697) Google Scholar). In the we found that the active of βarr1 allosterically activates C-Raf. Both the and of the complex the of C-Raf. that the interaction between βarr1 and the phosphorylated GPCR C terminus is sufficient to activate C-Raf. The by which a GPCR–βarr complex activates C-Raf the of proteins facilitate signal transduction through tethering signaling components into studies reveal that scaffold proteins allosterically regulate molecules in addition to tethering (22Pakharukova N. Masoudi A. Pani B. Staus D.P. Lefkowitz R.J. Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.J. Biol. Chem. 2020; 295: 16773-16784Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Yang F. Xiao P. Qu C.X. Liu Q. Wang L.Y. Liu Z.X. He Q.T. Liu C. Xu J.Y. Li R.R. Li M.J. Li Q. Guo X.Z. Yang Z.Y. He D.F. et al.Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.Nat. Chem. Biol. 2018; 14: 876-886Crossref PubMed Scopus (44) Google Scholar, M. G. J. A. The scaffold signaling by the MAP kinase for 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). In the we found that in addition to the canonical GPCR–βarr complexes also allosterically activate C-Raf. GPCR–βarr complexes the canonical scaffolding and allosteric roles by with the amino terminus of C-Raf. The canonical scaffolding and allosteric processes are and tethering and allosteric GPCR–βarr complexes the and of βarr-dependent MAPK signaling. Furthermore, GPCR–βarr complexes initiate the MAPK signaling through a and activate a MAPK is that GPCR–βarr complexes may allosterically activate other components in the MAPK cascade. that the active of βarr1 interacts with the regulatory region of C-Raf and allosterically the catalytic of C-Raf. The with C-terminal by a has been D.P. L.M. M. Pani B. A. Lefkowitz R.J. GPCR to reveal in beta-arrestin Natl. Acad. Sci. U. S. A. 2018; PubMed Scopus Google Scholar). C-Raf and terminus into with an terminus and C-terminal βarr1 into The and D.P. H. M.J. L.M. Lefkowitz R.J. Skiniotis G. Structure of the complex in a 2020; PubMed Scopus Google Scholar, M. A. S. S.S. S. synthetic to proteins in PubMed Scopus Google Scholar). H-Ras was a human C-Raf and terminus and as E. S. Li K. H. J.A. H. M.J. of and active 2019; PubMed Scopus Google Scholar). human C-Raf or terminus with an in The proteins and on the was by C-Raf was by terminus, and in and on by using and of S.S. M. E.M. A. Allosteric of using effector Struct. Mol. Biol. 2011; PubMed Scopus Google H-Ras R. I. K. R. B. M.S. M. G. J. and of directly regulate Full Text Full Text PDF PubMed Scopus Google and βarr1 and its Xiao K. Lefkowitz R.J. The active of evidence for the in the and in the active of and Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google have been and of with C-terminal by a was performed as D.P. L.M. M. Pani B. A. Lefkowitz R.J. GPCR to reveal in beta-arrestin Natl. Acad. Sci. U. S. A. 2018; PubMed Scopus Google Scholar). The synthetic phosphopeptide was on the C terminus of by as D.P. L.M. M. Pani B. A. Lefkowitz R.J. GPCR to reveal in beta-arrestin Natl. Acad. Sci. U. S. A. 2018; PubMed Scopus Google Scholar). The of was performed as D.P. H. M.J. L.M. Lefkowitz R.J. Skiniotis G. Structure of the complex in a 2020; PubMed Scopus Google Scholar). In was with excess for was with a of with and on for to the and the was was by and C-Raf by complexes or activated H-Ras was with a In C-Raf was with as a in the with the of was to initiate the of was to the then the of of was The of βarr1 or complexes in all was was with V2Rpp and for or other C-Raf then to the and for of and for with The and three using the proteins in and reduced and by using and The kinase of C-Raf was with To C-Raf kinase in C-Raf protein was with and for of into was by and data are J. Lefkowitz The that have of with the of We are to and for and Li for N. Y. A. and R. J. L. Y. Y. Y. R. J. L. data Y. and R. J. L. N. Y. A. and R. J. L. and Y. R. J. L. R. J. L. was by the of R. J. R. J. L. is a The is the of the and does not the of the of

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