Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Liang, Yue; Khoshouei, Maryam; Deganutti, Giuseppe; Glukhova, Alisa; Koole, Cassandra; Peat, Thomas S.; Radjainia, Mazdak; Plitzko, Jürgen M.; Baumeister, Wolfgang; Miller, Laurence J.; Hay, Debbie L.; Christopoulos, Arthur; Reynolds, Christopher A.; Wootten, Denise; Sexton, Patrick M.

doi:10.1038/s41586-018-0535-y

Cited by 224 publications

(380 citation statements)

References 66 publications

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“…These structures thus show the peptide to be bound as a linear α-helix with its Cterminal portion docked to the ECD and its N-terminal portion projecting into the core of the TMD bundle. This binding mode is similar to that seen in the high-resolution structures reported for several other family B GPCRs, including the receptors for glucagon, GLP-1, and calcitonin, each in complex with a bound peptide ligand, (14)(15)(16)(17)(18) thus supporting a common basic mechanism of ligand-induced activation used by each of the family B GPCRs.…”

Section: Introductionsupporting

confidence: 78%

“…1A. In functional studies in vitro, [Leu 11 ,dTrp 12 ,Trp 23 ,Tyr 36 ]-PTHrP(7-36)NH 2 was more effective as an inverse agonist than the PTH-based analog, [Nle 8,18 ,dTrp 12 , Tyr 34 ]-bPTH(7-34)NH 2, (24,25) because it more effectively reduced basal cAMP signaling in HEK293 cells stably transfected to express PTHR1-H223R and the GloSensor cAMP reporter ( Fig. 1B,C).…”

Section: Inverse Agonist Properties In Vitromentioning

confidence: 99%

“…PTHR1 is a family B G-protein-coupled receptor (GPCR) and, as for each of these receptors, binds its agonist peptide ligands, such as the bioactive PTH(1-34) fragment, via a two-site mechanism. This mechanism involves an initial docking of the (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) portion of the ligand to the amino-terminal extracellular domain (ECD) portion of the receptor and a subsequent engagement of the N-terminal (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) portion of the ligand with the extracellular loop and seven-transmembrane helical domain (TMD) region of the receptor, which leads to receptor activation. (10,11) The recent high-resolution X-ray crystal (12) and cryogenic electron microscopy (cryo-EM) (13) structures of the PTHR1, each in complex with a PTH or PTHrP analog ligand, confirm and extend this model of ligand binding.…”

Section: Introductionmentioning

confidence: 99%

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An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

Noda

Guo

Khatri

et al. 2019

Journal of Bone and Mineral Research

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Jansen's metaphyseal chondrodysplasia (JMC) is a rare disease of bone and mineral ion physiology that is caused by activating mutations in PTHR1. Ligand‐independent signaling by the mutant receptors in cells of bone and kidney results in abnormal skeletal growth, excessive bone turnover, and chronic hypercalcemia and hyperphosphaturia. Clinical features further include short stature, limb deformities, nephrocalcinosis, and progressive losses in kidney function. There is no effective treatment option available for JMC. In previous cell‐based assays, we found that certain N‐terminally truncated PTH and PTHrP antagonist peptides function as inverse agonists and thus can reduce the high rates of basal cAMP signaling exhibited by the mutant PTHR1s of JMC in vitro. Here we explored whether one such inverse agonist ligand, [Leu11,dTrp12,Trp23,Tyr36]‐PTHrP(7‐36)NH2 (IA), can be effective in vivo and thus ameliorate the skeletal abnormalities that occur in transgenic mice expressing the PTHR1‐H223R allele of JMC in osteoblastic cells via the collagen‐1α1 promoter (C1HR mice). We observed that after 2 weeks of twice‐daily injection and relative to vehicle controls, the IA analog resulted in significant improvements in key skeletal parameters that characterize the C1HR mice, because it reduced the excess trabecular bone mass, bone marrow fibrosis, and levels of bone turnover markers in blood and urine. The overall findings provide proof‐of‐concept support for the notion that inverse agonist ligands targeted to the mutant PTHR1 variants of JMC can have efficacy in vivo. Further studies of such PTHR1 ligand analogs could help open paths toward the first treatment option for this debilitating skeletal disorder. © 2019 American Society for Bone and Mineral Research.

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Section: Introductionsupporting

confidence: 78%

Section: Inverse Agonist Properties In Vitromentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

Noda

Guo

Khatri

et al. 2019

Journal of Bone and Mineral Research

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“…Furthermore, the potency of [TAMRA] 3 ‐hαCGRP 3 at both receptors was approximately equal to that of hαCGRP with no significant losses in potency, indicating that the addition of a fluorophore moiety is well‐tolerated at position 3 by CGRP. In the cryo‐EM structure of the peptide‐bound CGRP receptor, the side‐chain of Asp‐3 appears to reside in an exposed area, providing a possible explanation for the potent activity of this analogue . The structure of the AMY 1 receptor is currently unknown, although substitution of the asparagine at position 3 of human amylin with alanine is without effect .…”

Section: Resultsmentioning

confidence: 99%

A potent fluorescent calcitonin gene‐related peptide analogue enables visualization of receptor internalization

et al. 2019

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The calcitonin gene‐related peptide (CGRP) system provides a significant opportunity for the treatment of migraine. However, further understanding of the function of CGRP receptors is required. Fluorescent ligands are valuable probes that enable deeper understanding of peptide‐receptor interactions and receptor function. We herein report the synthesis and biological activity of a potent fluorescent CGRP that allowed direct observation of CGRP receptor internalization in a transfected cell system. The potent activity of this fluorescent CGRP thus enables access to a valuable pharmacological tool for further understanding the CGRP system.

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“…This residue is highly conserved throughout the peptide family (Thr 6 in AMY and CGRP, Thr 14 in IMD), and its particular importance for receptor activation has been highlighted by several studies . In fact, a recently published structure of CGRP bound to the active‐state CGRPR revealed a functionally important contact between the corresponding Thr 6 of CGRP and His 295 of the CLR . A similar function of ADM Thr 20 is very likely.…”

Section: Discussionmentioning

confidence: 99%

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM₁ receptor activation

Fischer

Schönauer

Els‐Heindl

et al. 2019

Journal of Peptide Science

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Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor‐like receptor (CLR), a class B G protein‐coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM‐1 receptor (AM1R). A disulfide‐bonded ring structure consisting of six amino acids between Cys16 and Cys21 has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t‐Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam‐based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether‐based mimetics to be well accepted with full AM1R activity. While a reduced selectivity over the calcitonin gene‐related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild–type‐like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM1R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.

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Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Cited by 224 publications

References 66 publications

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

A potent fluorescent calcitonin gene‐related peptide analogue enables visualization of receptor internalization

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM₁ receptor activation

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Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Cited by 224 publications

References 66 publications

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

A potent fluorescent calcitonin gene‐related peptide analogue enables visualization of receptor internalization

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM1 receptor activation

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Product

Resources

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Adrenomedullin disulfide bond mimetics uncover structural requirements for AM₁ receptor activation