Photo‐Cross‐Linkers Incorporated into G‐Protein‐Coupled Receptors in Mammalian Cells: A Ligand Comparison

Coin, Irene; Perrin, Marilyn H.; Vale, Wylie; Wang, Lei

doi:10.1002/ange.201102646

Cited by 19 publications

(10 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, because of the prolonged time scale of UV irradiation (seconds), PLC may trap transient or intermediate contacts unrepresentative of the ground-state structure (23). Nonstandard Pap mutagenesis (11), nonetheless, provides a general approach to characterize membrane proteins as exemplified by studies of intracellular protein docking at the EGF receptor (12) and ligand binding by G protein-coupled receptors (10,11).…”

Section: Discussionmentioning

confidence: 99%

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase

Whittaker¹,

Whittaker²,

Roberts

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The primary hormone-binding surface of the insulin receptor spans one face of the N-terminal β-helix of the α-subunit (the L1 domain) and an α-helix in its C-terminal segment (αCT). Crystallographic analysis of the free ectodomain has defined a contiguous dimerrelated motif in which the αCT α-helix packs against L1 β-strands 2 and 3. To relate structure to function, we exploited expanded genetic-code technology to insert photo-activatable probes at key sites in L1 and αCT. The pattern of αCT-mediated photocross-linking within the free and bound receptor is in accord with the crystal structure and prior mutagenesis. Surprisingly, L1 photoprobes in β-strands 2 and 3, predicted to be shielded by αCT, efficiently cross-link to insulin. Furthermore, anomalous mutations were identified on neighboring surfaces of αCT and insulin that impair hormone-dependent activation of the intracellular receptor tyrosine kinase (contained within the transmembrane β-subunit) disproportionately to their effects on insulin binding. Taken together, these results suggest that αCT, in addition to its hormonerecognition role, provides a signaling element in the mechanism of receptor activation.nonstandard mutagenesis | alanine scanning | affinity I nsulin plays a central role in the control of vertebrate metabolism. Despite its long-standing use in the treatment of diabetes mellitus, how insulin binds to and activates the insulin receptor (IR) poses a major unsolved problem (1). Recent advances in the structural characterization of the IR ectodomain have provided a foundation for reinvestigation of this classic problem (2, 3). Here, we have used structure-based photo-crosslinking (PCL) and mutagenesis to identify a dynamic signaling element at the hormone-binding surface of the ectodomain.Studies of insulin derivatives containing photo-activatable substitutions (para-azido-Phe; Pap) suggested that insulin undergoes a change in conformation on IR binding (4), partially exposing the hydrophobic core on displacement of the C-terminal segment of the B-chain (5). Such induced fit expands its nonpolar contact surface (1, 6). We hypothesized that induced fit of the IR could likewise expand its contact surface and in turn trigger receptor tyrosine kinase (TK) activation. Our studies focused on a tandem hormone-binding element comprising dimer-related structural motifs in the N-terminal β-helix of the α-subunit (L1 domain residues 1-158) and its C-terminal segment (αCT residues 704-715) (7,8). Introduction of Pap was accomplished by orthogonal tRNA/amber suppression (9). Guided by PCL, we have characterized a unique class of mutations in insulin and the IR ectodomain that impair TK activation disproportionately to effects on binding. Taken together, our results identify cognate recognition α-helices in insulin and its receptor that participate in transmission of the insulin signal.Our strategy had three parts. First, photo-probes were introduced by nonstandard mutagenesis (9-12) based on an ectodomain crystal structure (2, 3) and prior Ala scanning...

show abstract

Section: Discussionmentioning

confidence: 99%

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase

Whittaker¹,

Whittaker²,

Roberts

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Genetically encoded crosslinkers [30] have been widely applied to investigate peptide-GPCR interactions [31] both at rhodopsin-like [32,33] and secretin-like GPCRs. [25,34] Most recently,the method has been expanded to study GPCR-arrestin interactions [35] and GPCR oligomerization. [36] Photo-crosslinking mapping of Y 5 Rw ith Azi yielded crosslinking hits in three well-defined regions of the receptor:T he N-terminus,ECL2, and ECL3.…”

Section: Discussionmentioning

confidence: 99%

Binding of Natural Peptide Ligands to the Neuropeptide Y₅ Receptor

Rudolf

Kaempf

et al. 2021

Angewandte Chemie

Self Cite

View full text Add to dashboard Cite

The binding mode of natural peptide ligands to the Y 5 Gprotein-coupled receptor (Y 5 R), an attractive therapeutic target for the treatment of obesity,islargely unknown. Here,we apply complementary biochemical and computational approaches,i ncluding scanning of the receptor surface with ag enetically encoded crosslinker,A la-scanning of the ligand and double-cycle mutagenesis,t om ap interactions in the ligand-receptor interface and build as tructural model of the NPY-Y 5 Rc omplex guided by the experimental data. In the model, the carboxyl (C)-terminus of bound NPY is placed close to the extracellular loop (ECL) 3, whereas the characteristic a-helical segment of the ligand drapes over ECL1 and is tethered towards ECL2 by ah ydrophobic cluster.W ef urther show that the other two natural ligands of Y 5 R, peptide YY (PYY) and pancreatic polypeptide (PP) dock to the receptor in asimilar pose.

show abstract

“…F fact has been used to probe how a class B GPCR, corticotropin releasing factor receptor type 1 (CRF1R), interacts with its peptide ligand in mammalian cells (Figure 4). 9 To determine what sites of CRF1R interact with its native peptide ligand Urocortin‐1 (Ucn1), the photo‐cross‐linking Uaa p ‐azido‐phenylalanine (Azi) was first incorporated into various positions of CRF1R expressed in HEK293T cells to crosslink Ucn1 upon UV light activation 9,33 . Detection of the covalent complex of CRF1R‐Ucn1 in Western analysis of the cell lysate indicates that Ucn1 interacts with CRF1R at the Azi incorporation site.…”

Section: Covalent Bonding Between Two Proteinsmentioning

confidence: 99%

New covalent bonding ability for proteins

Cao

Wang

2021

Protein Science

Self Cite

View full text Add to dashboard Cite

To expand protein's covalent bonding ability, latent bioreactive unnatural amino acids have been designed and genetically encoded into proteins, which react with specific natural amino acid residues through proximity‐enabled bioreactivity. The resultant new covalent bonds can be selectively created within and between proteins in vitro, in cells, and in vivo. Offering diverse properties previously unattainable, these covalent linkages have been harnessed to enhance protein properties, to modulate protein function, to probe ligand–receptor binding, to identify elusive protein interactions, and to develop covalent protein drugs. Selective introduction of covalent bonds into proteins is affording novel avenues for biological studies, synthetic biology, and biotherapeutics.

show abstract

Photo‐Cross‐Linkers Incorporated into G‐Protein‐Coupled Receptors in Mammalian Cells: A Ligand Comparison

Cited by 19 publications

References 36 publications

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase

Binding of Natural Peptide Ligands to the Neuropeptide Y₅ Receptor

New covalent bonding ability for proteins

Contact Info

Product

Resources

About

Photo‐Cross‐Linkers Incorporated into G‐Protein‐Coupled Receptors in Mammalian Cells: A Ligand Comparison

Cited by 19 publications

References 36 publications

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase

α-Helical element at the hormone-binding surface of the insulin receptor functions as a signaling element to activate its tyrosine kinase

Binding of Natural Peptide Ligands to the Neuropeptide Y5 Receptor

New covalent bonding ability for proteins

Contact Info

Product

Resources

About

Binding of Natural Peptide Ligands to the Neuropeptide Y₅ Receptor