Identification of the Key Residue of Calcitonin Gene Related Peptide (CGRP) 27−37 to Obtain Antagonists with Picomolar Affinity at the CGRP Receptor

Lang, Manja; Pol, Silvia De; Baldauf, Carsten; Hofmann, Hans‐Jörg; Reiser, Oliver; Beck‐Sickinger, Annette G.

doi:10.1021/jm050613s

Cited by 38 publications

(52 citation statements)

References 45 publications

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“…A β -turn centered on P34 has been shown to be important for the activity of the related calcitonin60,71,72 and CGRP peptides 62,73,74. Similar β -turns have been shown to be a common feature for the activation of other peptide-activated G-coupled protein receptors 75.…”

Section: Discussionmentioning

confidence: 96%

Three-Dimensional Structure and Orientation of Rat Islet Amyloid Polypeptide Protein in a Membrane Environment by Solution NMR Spectroscopy

et al. 2009

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Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide hormone associated with glucose metabolism that is cosecreted with insulin by β-cells in the pancreas. Since human IAPP is a highly amyloidogenic peptide, it has been suggested that the formation of IAPP amyloid fibers is responsible for the death of β-cells during the early stages of type II diabetes. It has been hypothesized that transient membrane-bound α-helical structures of human IAPP are precursors to the formation of these amyloid deposits. On the other hand, rat IAPP forms transient α-helical structures but does not progress further to form amyloid fibrils. To understand the nature of this intermediate state and the difference in toxicity between the rat and human versions of IAPP, we have solved the high-resolution structure of rat IAPP in the membrane-mimicking detergent micelles composed of dodecylphosphocholine. The structure is characterized by a helical region spanning the residues A5 to S23 and a disordered C-terminus. A distortion in the helix is seen at R18 and S19 that may be involved in receptor binding. Paramagnetic quenching NMR experiments indicate that rat IAPP is bound on the surface of the micelle, in agreement with other nontoxic forms of IAPP. A comparison to the detergent-bound structures of other IAPP variants indicates that the N-terminal region may play a crucial role in the self-association and toxicity of IAPP by controlling access to the putative dimerization interface on the hydrophobic face of the amphipathic helix.

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Section: Discussionmentioning

confidence: 96%

Three-Dimensional Structure and Orientation of Rat Islet Amyloid Polypeptide Protein in a Membrane Environment by Solution NMR Spectroscopy

et al. 2009

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“…There has been some work on the structure–activity relationship for the C‐terminus of CGRP acting on the CGRP receptor (Conner et al ., ; Lang et al ., ), but much less is known about the N‐terminus of the peptide when it activates this receptor or the AMY 1 receptor (Watkins et al ., ). The importance of the disulphide bond has been established (Dennis et al ., ; Saha et al ., ), and there are a few studies based mainly on deletions (Thiebaud et al ., ; Hakala et al ., ; Heino et al ., ).…”

Section: Introductionmentioning

confidence: 99%

Structure–activity relationships of the N‐terminus of calcitonin gene‐related peptide: key roles of alanine‐5 and threonine‐6 in receptor activation

Hay

Harris

Kowalczyk

et al. 2013

British J Pharmacology

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BACKGROUND AND PURPOSEThe N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. EXPERIMENTAL APPROACHCGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. KEY RESULTSSubstitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. CONCLUSIONS AND IMPLICATIONSAla-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

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“…While the α-helix of human CGRP is also similar to that of Aaeg -DH 31 , homologous key residues for receptor activation were not found in Aaeg -DH 31 (Fig. S4B) [34]. In contrast, the Aaeg -DH 31 C-terminal amidated proline (Fig.…”

Section: Resultsmentioning

confidence: 84%

Role in Diuresis of a Calcitonin Receptor (GPRCAL1) Expressed in a Distal-Proximal Gradient in Renal Organs of the Mosquito Aedes aegypti (L.)

Kwon

Longnecker

et al. 2012

PLoS ONE

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Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na+, Cl−) is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH31) are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL) in the mosquito Malpighian tubules (MTs), the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH31 receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH31. Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs arose early in evolution.

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Identification of the Key Residue of Calcitonin Gene Related Peptide (CGRP) 27−37 to Obtain Antagonists with Picomolar Affinity at the CGRP Receptor

Cited by 38 publications

References 45 publications

Three-Dimensional Structure and Orientation of Rat Islet Amyloid Polypeptide Protein in a Membrane Environment by Solution NMR Spectroscopy

Three-Dimensional Structure and Orientation of Rat Islet Amyloid Polypeptide Protein in a Membrane Environment by Solution NMR Spectroscopy

Structure–activity relationships of the N‐terminus of calcitonin gene‐related peptide: key roles of alanine‐5 and threonine‐6 in receptor activation

Role in Diuresis of a Calcitonin Receptor (GPRCAL1) Expressed in a Distal-Proximal Gradient in Renal Organs of the Mosquito Aedes aegypti (L.)

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