scholarly journals Optimization of a peptide ligand for the adhesion GPCR ADGRG2 provides a potent tool to explore receptor biology

2020 ◽  
pp. jbc.RA120.014726
Author(s):  
Yujing Sun ◽  
Daolai Zhang ◽  
Ming-Liang Ma ◽  
Hui Lin ◽  
Youchen Song ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Unnatural Amino Acid ◽  
Peptide Ligand ◽  
Toggle Switch ◽  
Ph Homeostasis ◽  
Alanine Scanning Mutagenesis ◽  
Critical Residues ◽  
Receptor Biology ◽  
Peptide Agonist ◽  
Adhesion Gpcr

The adhesion GPCR ADGRG2, also known as GPR64, is a critical regulator of male fertility that maintains ion/pH homeostasis and CFTR coupling. The molecular basis of ADGRG2 function is poorly understood, in part due to the fact that no endogenous ligands for ADGRG2 have been reported, thus limiting the tools available to interrogate ADGRG2 activity. It has been shown that ADGRG2 can be activated by a peptide, termed p15, derived from its own N-terminal region known as the Stachel sequence. However, the low affinity of p15 limits its utility for ADGRG2 characterization. In the current study, we used alanine scanning mutagenesis to examine the critical residues responsible for p15-induced ADGRG2 activity. We next designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural and unnatural amino acid substitutions. We obtained an optimized ADGRG2 Stachel peptide T1V/F3Phe(4-Me) (VPM-p15) that activated ADGRG2 with significantly improved (>2 orders of magnitude) affinity. We then characterized the residues in ADGRG2 that were important for ADGRG2 activation in response to VPM-p15 engagement, finding that the toggle switch W6.53 and residues of ECL2 region of ADGRG2 are key determinants for VPM-p15 interactions and VPM-p15 induced Gs or arrestin signaling. Our study not only provides a useful tool to investigate the function of ADGRG2, but also offers new insights to guide further optimization of Stachel peptides to activate adhesion GPCR members.

scholarly journals Constitutive signaling by the C-terminal fragment of polycystin-1 is mediated by a tethered peptide agonist

2021 ◽  
Author(s):  
Brenda S Magenheimer ◽  
Ericka Nevarez Munoz ◽  
Jayalakshmi Ravichandran ◽  
Robin L Maser
Keyword(s):  
G Protein ◽  
Transmembrane Domain ◽  
Missense Mutations ◽  
Protein Signaling ◽  
Gene Encoding ◽  
Reporter Assays ◽  
Autosomal Dominant Polycystic Kidney ◽  
Signaling Activation ◽  
Peptide Agonist ◽  
Adhesion Gpcr

Mutation of the PKD1 gene, encoding polycystin-1 (PC1), is the primary cause of autosomal dominant polycystic kidney disease. PC1 is an 11-transmembrane domain protein that binds and modulates the activity of multiple heterotrimeric G protein families and is thought to function as a non-canonical G protein-coupled receptor (GPCR). PC1 shares a conserved GPCR autoproteolysis inducing (GAIN) domain with the adhesion family of GPCRs, that promotes an auto-catalytic, cis-cleavage at the GPCR proteolysis site (GPS) located proximal to the first transmembrane domain. GPS cleavage divides these receptors into two associated subunits, the extracellular N-terminal (NTF) and transmembrane C-terminal (CTF) fragments. For the adhesion GPCRs, removal of the NTF leads to activation of G protein signaling as a result of the exposure and subsequent intramolecular binding of the extracellular N-terminal stalk of the CTF, i.e., the tethered cryptic ligand or tethered agonist model. Here, we test the hypothesis that PC1-mediated signaling is regulated by an adhesion GPCR-like, tethered agonist mechanism. Using cell-based reporter assays and mutagenesis of PC1 expression constructs, we show that the CTF form of PC1 requires the stalk for signaling activation and synthetic peptides derived from the PC1 stalk sequence can re-activate signaling by a stalk-less CTF. In addition, we demonstrate that ADPKD-associated missense mutations within the PC1 stalk affect signaling and can inhibit GPS cleavage. These results provide a foundation for beginning to understand the molecular mechanism of G protein regulation by PC1 and suggest that a tethered agonist-mediated mechanism can contribute to PKD pathogenesis.

scholarly journals Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 507 ◽  
Author(s):  
Jiong Ning ◽  
Rui Li ◽  
Jie Ren ◽  
Dongting Zhangsun ◽  
Xiaopeng Zhu ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
The Novel ◽  
Muscle Type ◽  
Alanine Scanning Mutagenesis ◽  
Dynamic Simulations ◽  
Alanine Scanning ◽  
Critical Residue ◽  
Hydrophobic Amino Acids ◽  
Critical Residues

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure–activity relationship (SAR) between GI and mouse α1β1δε nAChRs. The Pro5, Gly8, Arg9, and Tyr11 were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1β1δε nAChR. On the contrary, substituting Asn4, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1β1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1β1δε nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse α1β1δε nAChR in detail that will help to develop the novel analogues of GI.

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

2002 ◽  
Vol 80 (5) ◽  
pp. 413-417 ◽  
Author(s):  
Claudio M Costa-Neto ◽  
Ayumi A Miyakawa ◽  
João B Pesquero ◽  
Laerte Oliveira ◽  
Siv A Hjorth ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Peptide Ligand ◽  
Wild Type ◽  
Angiotensin Ii Receptor ◽  
Extracellular Region ◽  
Preferential Binding ◽  
Peptide Agonist ◽  
Mutant Forms

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G196I, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G196I mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists. Key words: angiotensin, receptor, GPCR, non-peptide agonist, transduction.

scholarly journals Molecular Basis for T Cell Response Induced by Altered Peptide Ligand of Type II Collagen

2012 ◽  
Vol 287 (23) ◽  
pp. 19765-19774 ◽  
Author(s):  
Jeoung-Eun Park ◽  
David Cullins ◽  
Lillian Zalduondo ◽  
Stacey L. Barnett ◽  
Ae-Kyung Yi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Molecular Basis ◽  
Type Ii Collagen ◽  
T Cell Response ◽  
Peptide Ligand ◽  
Altered Peptide Ligand ◽  
Type Ii

scholarly journals Alanine-scanning Mutagenesis of Plasmatocyte Spreading Peptide Identifies Critical Residues for Biological Activity

2001 ◽  
Vol 276 (21) ◽  
pp. 18491-18496 ◽  
Author(s):  
Kevin D. Clark ◽  
Brian F. Volkman ◽  
Honglada Thoetkiattikul ◽  
David King ◽  
Yoichi Hayakawa ◽  
...  
Keyword(s):  
Biological Activity ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Critical Residues

scholarly journals Novel septin 9 repeat motifs altered in neuralgic amyotrophy bind and bundle microtubules

2013 ◽  
Vol 203 (6) ◽  
pp. 895-905 ◽  
Author(s):  
Xiaobo Bai ◽  
Jonathan R. Bowen ◽  
Tara K. Knox ◽  
Kaifeng Zhou ◽  
Manuela Pendziwiat ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Autosomal Dominant ◽  
Neurite Growth ◽  
The Novel ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Neuralgic Amyotrophy ◽  
Septin 9 ◽  
Insight Into ◽  
Β Tubulin

Septin 9 (SEPT9) interacts with microtubules (MTs) and is mutated in hereditary neuralgic amyotrophy (HNA), an autosomal-dominant neuropathy. The mechanism of SEPT9 interaction with MTs and the molecular basis of HNA are unknown. Here, we show that the N-terminal domain of SEPT9 contains the novel repeat motifs K/R-x-x-E/D and R/K-R-x-E, which bind and bundle MTs by interacting with the acidic C-terminal tails of β-tubulin. Alanine scanning mutagenesis revealed that the K/R-R/x-x-E/D motifs pair electrostatically with one another and the tails of β-tubulin, enabling septin–septin interactions that link MTs together. SEPT9 isoforms lacking repeat motifs or containing the HNA-linked mutation R88W, which maps to the R/K-R-x-E motif, diminished intracellular MT bundling and impaired asymmetric neurite growth in PC-12 cells. Thus, the SEPT9 repeat motifs bind and bundle MTs, and thereby promote asymmetric neurite growth. These results provide the first insight into the mechanism of septin interaction with MTs and the molecular and cellular basis of HNA.

scholarly journals Identification of a novel thrombin receptor sequence required for activation-dependent responses

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4195-4202 ◽  
Author(s):  
WF Bahou ◽  
JL Kutok ◽  
A Wong ◽  
CL Potter ◽  
BS Coller
Keyword(s):  
Xenopus Oocytes ◽  
Epitope Mapping ◽  
Cell Types ◽  
Receptor Activation ◽  
Peptide Ligands ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombin Receptor ◽  
Peptide Ligand ◽  
Amino Acid Residues ◽  
Critical Residues

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1–160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34–44, 48–67, 65–79, and 87–94. Soluble peptides derived from regions 83–94, but not those from other regions of the receptor, neutralized the ability of anti-TR1–160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67–82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42–47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N- terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.

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