scholarly journals Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors

2020 ◽  
Author(s):  
Jason M. Booe ◽  
Margaret L. Warner ◽  
Augen A. Pioszak
Keyword(s):  
Drug Targets ◽  
Rational Design ◽  
Transmembrane Domain ◽  
Calcitonin Gene Related Peptide ◽  
Peptide Ligands ◽  
Ligand Selectivity ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Receptor Activity Modifying Proteins ◽  
Long Acting

AbstractThe calcitonin receptor-like G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) actions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) determine CLR ligand selectivity through an unresolved mechanism. CLR-RAMP complexes are drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. We used combinatorial peptide library and rational design approaches to probe selectivity determinants and develop short AM and CGRP variants with ∼1000-fold increased receptor extracellular domain affinities. Binding and structural studies explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in selectivity. In longer scaffolds that also bind the CLR transmembrane domain the variants generated picomolar affinity antagonists, one with an estimated 12.5 hr CGRP receptor residence time, and sustained signaling agonists ss-AM and ss-CGRP. This work clarifies the RAMP-modulated ligand selectivity mechanism and provides AM and CGRP variants with promise as long-acting therapeutics.

scholarly journals Overview of Calcitonin Gene-Related Peptide and Its Receptor

2001 ◽  
Vol 1 ◽  
pp. 1-1 ◽  
Author(s):  
Nambi Aiyar
Keyword(s):  
Transmembrane Domain ◽  
Biological Activities ◽  
Cardiovascular Effects ◽  
Functional Expression ◽  
Calcitonin Gene Related Peptide ◽  
Structural Modifications ◽  
Amino Acid Peptide ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
C And N

Calcitonin Gene-Related Peptide (CGRP), a 37 amino acid peptide identified as the alternately spliced gene product of calcitonin gene, is a sensory neuropeptide with potent cardiovascular effects. CGRP is distributed throughout the central and peripheral nervous systems and possesses diverse biological actions. CGRP has been suggested to play a role in diseases such as migraine, diabetes, pain, and inflammation. Two forms of CGRP (alpha and beta) that differ in three amino acids have been identified and are encoded by different genes. Based on the differential biological activities of various CGRP analogs, the CGRP receptors have been classified into CGRP1 and CGRP2. Structure-activity studies of CGRP analogs showed that the C- and N-terminal regions of the peptide interact independently with their receptors. While C-terminal peptide, CGRP (8-37) behaves as a CGRP1 receptor antagonist, N-terminal peptide CGRP (1-12) behaves as a weak agonist. Structural modifications of CGRP(28-37) have yielded micromolar to nanomolar affinity ligands. CGRP receptor belongs to the calcitonin receptor like receptor (CRLR) family of G-protein-coupled receptors and has been shown to require a single transmembrane domain protein called receptor activity modifying protein-1 (RAMP1) for its functional expression as well as activity. Human, rat, and porcine CRLRs have been cloned and characterized. Currently, the major focus is on the identification of potent and specific nonpeptide antagonists for this receptor in order to understand the physiological and pathophysiological role of this peptide.

scholarly journals Biochemical characterization of G protein coupling to calcitonin gene–related peptide and adrenomedullin receptors using a native PAGE assay

2020 ◽  
Vol 295 (28) ◽  
pp. 9736-9751 ◽  
Author(s):  
Amanda M. Roehrkasse ◽  
Margaret L. Warner ◽  
Jason M. Booe ◽  
Augen A. Pioszak
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Calcitonin Gene Related Peptide ◽  
Peptide Ligand ◽  
Native Page ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
Ligand Selectivity ◽  
Related Peptide ◽  
Calcitonin Gene

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein–coupled receptor (CLR), which heterodimerizes with three receptor activity–modifying proteins (RAMP1–3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein–tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

scholarly journals CGRP Receptor Family and Accessory Protein Localization: Implications for Predicted Function

2001 ◽  
Vol 1 ◽  
pp. 10-10
Author(s):  
K.R. Oliver
Keyword(s):  
Protein Localization ◽  
Calcitonin Gene Related Peptide ◽  
Receptor Activity ◽  
Calcitonin Receptor ◽  
Cgrp Receptor ◽  
Related Peptide ◽  
Cellular Expression ◽  
Receptor Component ◽  
Calcitonin Gene ◽  
Receptor Activity Modifying Proteins

Calcitonin gene-related peptide (CGRP), adrenomedullin, amylin, and calcitonin are functionally related neuropeptides. Certain of these peptides mediate their action through receptors which have common components, such as the receptor activity modifying proteins (RAMPs) and CGRP-receptor component protein, as well as possibly through other distinct receptors. Specifically, the molecular pharmacology of CGRP and adrenomedullin is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Additionally, through formation of another hetero-oligomer, RAMPs also govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. We have used multiple approaches to discern the regional and cellular expression of these various receptor components and binding sites for the above neuropeptides in multiple species and in different tissues. Techniques applied include in situ hybridization, immunohistochemistry and radioligand autoradiography. These data allow further understanding of both the complexity of receptor-receptor component and receptor-ligand interactions in vivo. Interestingly, these localization data suggest that RAMPs may interact with receptors additional to those already identified for the CGRP family and may be involved in binding innate neuropeptides or other neurotransmitters which are not members of the calcitonin gene-related peptide fam

Sign in / Sign up

Export Citation Format

Share Document