Allosteric activation of proto-oncogene kinase Src by GPCR–beta-arrestin complexes

Pakharukova, Natalia; Masoudi, Ali; Pani, Biswaranjan; Staus, Dean P.; Lefkowitz, Robert J.

doi:10.1074/jbc.ra120.015400

Cited by 31 publications

(40 citation statements)

References 43 publications

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“…Many GPCRs are internalized into endosomes via a clathrin-dependent pathway and partly degraded in lysosomes or recycled to the cell membrane for prolonged agonist stimulation [ 36 , 37 , 38 ]. In the present study, the t 1/2 rate indicated slightly faster cell-surface receptor loss in cells expressing the L457R mutant than in cells expressing the wild-type receptor, whereas the rate of loss in the former was slower than for wild-type eLH/CGR, D564G, and D578Y after 30 min.…”

Section: Discussionmentioning

confidence: 99%

“…Research is currently underway to clarify our theory on GPCR internalization, degradation, and recycling. Recently, biased allosteric modulators and regulators of other GPCR have been reported [ 36 , 37 , 40 ]. In the present study, the mutants located in the transmembrane domain, and between intracellular loops and the transmembrane domain, demonstrated that eLH/CGRs may be involved in interactions with different types of G-proteins.…”

Section: Discussionmentioning

confidence: 99%

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Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Byambaragchaa

Seong

Choi

et al. 2021

IJMS

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The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Byambaragchaa

Seong

Choi

et al. 2021

IJMS

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“…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 function not just as typical scaffold proteins for the C-J o u r n a l P r e -p r o o f GPCR-βarr1 complexes allosterically activate C-Raf by interacting with its amino-terminus GPCR-βarr complexes have been demonstrated to allosterically activate signaling partners such as the tyrosine kinase Src (22,23). To investigate whether GPCR-βarr complexes allosterically regulate C-Raf activation, we used an enzyme-coupled fluorescence assay to measure the C-Raf activity in real time (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

Zang¹,

Kahsai²,

Pakharukova³

et al. 2021

Journal of Biological Chemistry

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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.

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“…leading to the adoption of an open active conformation of the kinase [ 76 ]. A further recent study verified that receptor-bound arrestin-2, but not free arrestin-2, is able to activate Src [ 43 ]. Here, the binding of the receptor phospho-tail to arrestin-2 was shown to be sufficient to activate arrestin-2 and therefore Src ( Figure 2 A).…”

Section: Src Activation Through a Gpcr–arrestin Complexmentioning

confidence: 96%

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation

Berndt

Liebscher

2021

IJMS

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Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR–SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

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Allosteric activation of proto-oncogene kinase Src by GPCR–beta-arrestin complexes

Cited by 31 publications

References 43 publications

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro

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

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation

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