The structure of secretin family GPCR peptide ligands: implications for receptor pharmacology and drug development

Watkins, Harriet A.; Au, Maggie; Hay, Debbie L.

doi:10.1016/j.drudis.2012.05.005

Cited by 49 publications

(64 citation statements)

References 71 publications

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“…Of interest, the position 10 probe did not yield any demonstrable disulfide bonds at all despite having binding affinity at least as high as the other probes and being fully biologically active. This is consistent with the side group of that residue establishing a hydrophobic interaction with the side group of residue five as part of the N-capping motif (1,23). It might, therefore, play a role in internal stabilization of such a motif rather than being available to interact directly with the FIGURE 6.…”

Section: Discussionsupporting

confidence: 60%

“…The helix N-capping motif that is highly conserved within the natural peptide ligands for the class B1 G protein-coupled receptors has been the focus of substantial interest in recent years (1,23). In addition to its likely role to stabilize the helical conformation extending through the mid-region and carboxylterminal region of these peptides, it has been postulated to play a direct role in activation of these receptors and possibly even to serve as a structural lead to small molecule agonist development (1, 23).…”

Section: Discussionmentioning

confidence: 99%

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Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Dong

Lam

Orry

et al. 2016

Journal of Biological Chemistry

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Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin (sec), Phe 6 , Thr 7 , and Leu 10 , and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys 6 -sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys 7 -sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys 10 -sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Dong

Lam

Orry

et al. 2016

Journal of Biological Chemistry

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“…Its main feature is its large N-terminal extracellular domain (ECD) (Watkins et al, 2012), which is crucial for the recognition and binding of ligands, typically peptides or hormones. Historically, this family was named for the intestinal hormone secretin, which, in the early 20th century, was the first hormone discovered (Bayliss and Starling, 1902).…”

Section: Gpcr Familiesmentioning

confidence: 99%

Outside-in signaling – a brief review of GPCR signaling with a focus on the Drosophila GPCR family

Hanlon

Andrew

2015

Journal of Cell Science

112

120

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G-protein-coupled receptors (GPCRs) are the largest family of receptors in many organisms, including worms, mice and humans. GPCRs are seven-transmembrane pass proteins that are activated by binding a stimulus (or ligand) in the extracellular space and then transduce that information to the inside of the cell through conformational changes. The conformational changes activate heterotrimeric G-proteins, which execute the downstream signaling pathways through the recruitment and activation of cellular enzymes. The highly specific ligand-GPCR interaction prompts an efficient cellular response, which is vital for the health of the cell and organism. In this Commentary, we review general features of GPCR signaling and then focus on the Drosophila GPCRs, which are not as wellcharacterized as their worm and mammalian counterparts. We discuss findings that the Drosophila odorant and gustatory receptors are not bona fide GPCRs as is the case for their mammalian counterparts. We also present here a phylogenetic analysis of the bona fide Drosophila GPCRs that suggest potential roles for several family members. Finally, we discuss recently discovered roles of GPCRs in Drosophila embryogenesis, a field we expect will uncover many previously unappreciated functions for GPCRs.

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“…20 CGRP and AM contain a conserved disulfide bond structure in their Nterminal regions that interact with the receptor helical bundle and variable sequence in their C-terminal regions that interact with the receptor ECDs. 21 It is unclear if CGRP and AM bind to their receptor ECDs as continuous a-helices as observed for other class B GPCR ECD-peptide pairs. Chimeric receptor and mutagenesis studies indicated that the RAMP ECDs are sufficient to determine peptide selectivity and identified receptor and CGRP and AM peptide residues that are important for their interactions, [22][23][24][25][26][27] but precisely how RAMPs determine peptide selectivity remains uncertain.…”

mentioning

confidence: 99%

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

Moad

Pioszak

2013

Protein Science

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Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.

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The structure of secretin family GPCR peptide ligands: implications for receptor pharmacology and drug development

Cited by 49 publications

References 71 publications

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Outside-in signaling – a brief review of GPCR signaling with a focus on the Drosophila GPCR family

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

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