Genetic code expansion and photocross-linking identify different β-arrestin binding modes to the angiotensin II type 1 receptor

Gagnon, Laurence; Cao, Yuan; Cho, Aaron; Sedki, Dana; Huber, Thomas; Sakmar, Thomas P.; Laporte, Stéphane A.

doi:10.1074/jbc.ra119.010324

Cited by 25 publications

(26 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To our knowledge, this was the first experimental evidence documenting an unnatural amino acid replacement in a GPCR expressed in its native environment, and the use of a mutated receptor to photocapture a peptide ligand. Similar UAAR experiments were carried out subsequently on other GPCRs [ 139 , 140 , 141 , 142 ].…”

Section: Genetic Manipulation Of the Receptor Structurementioning

confidence: 78%

A Paradigm for Peptide Hormone-GPCR Analyses

Naider

Becker

2020

Molecules

View full text Add to dashboard Cite

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

show abstract

Section: Genetic Manipulation Of the Receptor Structurementioning

confidence: 78%

A Paradigm for Peptide Hormone-GPCR Analyses

Naider

Becker

2020

Molecules

View full text Add to dashboard Cite

show abstract

“…The arr-FL of arrestin-1 assumes a helical structure in both the rhodopsin/arr-FL and rhodopsin/arrestin-1 crystal structures, but positioning of this motif differs significantly, suggesting that arrestin-1 may engage the rhodopsin in different conformations. Such conformational plasticity is further supported by a recent study using unnatural amino acid p-azido-L-phenylalanine incorporation into 25 different positions of cytosolic domains of AngII (angiotensin II) type 1 receptor (AT1R) and UV-induced photocross-linking with b-arrestins (122). The study revealed different interaction patterns between b-arrestins and AT1R, suggesting that AT1R/ b-arrestin complexes of different conformations may form.…”

Section: Paradoxical Actions Of B-arrestin As Terminator and Promotermentioning

confidence: 80%

Structural insights into emergent signaling modes of G protein–coupled receptors

Sutkevičiūtė

Vilardaga

2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

G protein–coupled receptors (GPCRs) represent the largest family of cell membrane proteins, with >800 GPCRs in humans alone, and recognize highly diverse ligands, ranging from photons to large protein molecules. Very important to human medicine, GPCRs are targeted by about 35% of prescription drugs. GPCRs are characterized by a seven-transmembrane α-helical structure, transmitting extracellular signals into cells to regulate major physiological processes via heterotrimeric G proteins and β-arrestins. Initially viewed as receptors whose signaling via G proteins is delimited to the plasma membrane, it is now recognized that GPCRs signal also at various intracellular locations, and the mechanisms and (patho)physiological relevance of such signaling modes are actively investigated. The propensity of GPCRs to adopt different signaling modes is largely encoded in the structural plasticity of the receptors themselves and of their signaling complexes. Here, we review emerging modes of GPCR signaling via endosomal membranes and the physiological implications of such signaling modes. We further summarize recent structural insights into mechanisms of GPCR activation and signaling. We particularly emphasize the structural mechanisms governing the continued GPCR signaling from endosomes and the structural aspects of the GPCR resensitization mechanism, and discuss the recently uncovered and important roles of lipids in these processes.

show abstract

“…Phosphorylation of the C-tail is known to increase b-arrestin affinity for GPCRs (18,32,33). Additionally, molecular and structural studies have established a role for the 8 th helix in enabling both visual arrestin and b-arrestin interactions with GPCRs (19,(34)(35)(36). Both of the motifs investigated in this study have been studied for PAR2 but in different cellular backgrounds (21)(22)(23).…”

Section: Discussionmentioning

confidence: 98%

Role of the C-terminal tail in regulating Proteinase Activated Receptor 2 (PAR2) signalling

Thibeault

Ramachandran

2020

Preprint

View full text Add to dashboard Cite

The C-terminal tail of G-proteincoupled receptors contain important regulatory sites that enable interaction with intracellular signalling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser 383-385 , Ser 387 -Thr 392 ) and the helix-8 palmitoylation site (Cys 361 ) in signalling regulation downstream of the proteolytically-activated GPCR, PAR2. We examined Gaq/11-coupled calcium signalling, b-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2knockout HEK-293 cell background. We find that alanine substitution of the membrane proximal serine residues (Ser 383-385 Ala) had no effect on SLIGRL-NH2-or trypsin-stimulated b-arrestin recruitment. Alanine substitutions in the Ser 387 -Thr 392 cluster resulted in a large (~50%) decrease in b-arrestin-1/2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without effect on trypsinactivated b-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys 361 Ala) led to a (~50%) decrease in b-arrestin-1/-2 recruitment in response to both trypsin and SLIGRL-NH2. We further show that Gaq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of b-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of b-arrestins also enhanced Gaq/11mediated calcium signalling. In line with these findings, C-tail serine/threonine and cysteine

show abstract

Genetic code expansion and photocross-linking identify different β-arrestin binding modes to the angiotensin II type 1 receptor

Cited by 25 publications

References 55 publications

A Paradigm for Peptide Hormone-GPCR Analyses

A Paradigm for Peptide Hormone-GPCR Analyses

Structural insights into emergent signaling modes of G protein–coupled receptors

Role of the C-terminal tail in regulating Proteinase Activated Receptor 2 (PAR2) signalling

Contact Info

Product

Resources

About