The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.

Download Full-text

The role of the extracellular loops of the CGRP receptor, a family B GPCR

Biochemical Society Transactions ◽

10.1042/bst20110726 ◽

2012 ◽

Vol 40 (2) ◽

pp. 433-437 ◽

Cited By ~ 13

Author(s):

James Barwell ◽

Michael J. Woolley ◽

Mark Wheatley ◽

Alex C. Conner ◽

David R. Poyner

Keyword(s):

Surface Expression ◽

Receptor Activation ◽

Receptor Expression ◽

Cell Surface Expression ◽

Calcitonin Receptor ◽

Transmembrane Helices ◽

Extracellular Loops ◽

Calcitonin Gene ◽

G Protein Coupled

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

Download Full-text

The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activlation

Frontiers in Endocrinology ◽

10.3389/fendo.2021.792912 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ian Winfield ◽

Kerry Barkan ◽

Sarah Routledge ◽

Nathan J. Robertson ◽

Matthew Harris ◽

...

Keyword(s):

Molecular Dynamics ◽

G Protein ◽

Conformational Changes ◽

Transmembrane Helix ◽

Receptor Activation ◽

Cgrp Receptor ◽

Intracellular Loop ◽

Glucagon Receptor ◽

G Protein Coupled

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

Download Full-text

Structure–function analyses reveal a triple β-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005062 ◽

2018 ◽

Vol 293 (41) ◽

pp. 15840-15854 ◽

Cited By ~ 10

Author(s):

Amanda M. Roehrkasse ◽

Jason M. Booe ◽

Sang-Min Lee ◽

Margaret L. Warner ◽

Augen A. Pioszak

Keyword(s):

Calcitonin Receptor ◽

Cgrp Receptor ◽

Ligand Selectivity ◽

Receptor Complexes ◽

Intramolecular Disulfide Bond ◽

Calcitonin Gene ◽

Class B ◽

Biased Signaling ◽

Receptor Pharmacology ◽

G Protein Coupled

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor–like class B G protein–coupled receptor (CLR) and a variable receptor activity–modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP–CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment–bound RAMP1–CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple β-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Download Full-text

The activation of the CGRP receptor

Biochemical Society Transactions ◽

10.1042/bst20120251 ◽

2013 ◽

Vol 41 (1) ◽

pp. 180-184 ◽

Cited By ~ 8

Author(s):

James Barwell ◽

Mark Wheatley ◽

Alex C. Conner ◽

Bruck Taddese ◽

Shabana Vohra ◽

...

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Accessory Protein ◽

Calcitonin Receptor ◽

Cgrp Receptor ◽

Peptide Receptor ◽

Calcitonin Gene ◽

New Knowledge ◽

G Protein Coupled

Download Full-text

Interaction of calcitonin-gene-related peptide with its receptors

Biochemical Society Transactions ◽

10.1042/bst0300451 ◽

2002 ◽

Vol 30 (4) ◽

pp. 451-455 ◽

Cited By ~ 37

Author(s):

A. C. Conner ◽

D. L. Hay ◽

S. G. Howitt ◽

K. Kilk ◽

Ü. Langel ◽

...

Keyword(s):

Direct Contact ◽

Transmembrane Domain ◽

Receptor Activation ◽

Calcitonin Gene Related Peptide ◽

Type Ii ◽

Calcitonin Receptor ◽

High Affinity ◽

Related Peptide ◽

Calcitonin Gene ◽

G Protein Coupled

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28–37 and 8–18 are normally required for high-affinity binding, while residues 19–27 form a hinge region. The 28–37 region is almost certainly in direct contact with the receptor; 8–18 may make additional receptor contacts or may stabilize an appropriate conformation of 28–37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.

Download Full-text

Ligand binding and activation of the CGRP receptor

Biochemical Society Transactions ◽

10.1042/bst0350729 ◽

2007 ◽

Vol 35 (4) ◽

pp. 729-732 ◽

Cited By ~ 20

Author(s):

A.C. Conner ◽

J. Simms ◽

J. Barwell ◽

M. Wheatley ◽

D.R. Poyner

Keyword(s):

G Protein ◽

Binding Pocket ◽

Receptor Activation ◽

Activation Mechanism ◽

Extracellular Loop ◽

C Terminus ◽

Calcitonin Gene ◽

The Family ◽

N Terminus ◽

G Protein Coupled

The receptor for CGRP (calcitonin gene-related peptide) is a heterodimer between a GPCR (G-protein-coupled receptor), CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). Models have been produced of RAMP1 and CLR. It is likely that the C-terminus of CGRP interacts with the extracellular N-termini of CLR and RAMP1; the extreme N-terminus of CLR is particularly important and may interact directly with CGRP and also with RAMP1. The N-terminus of CGRP interacts with the TM (transmembrane) portion of the receptor; the second ECL (extracellular loop) is especially important. Receptor activation is likely to involve the relative movements of TMs 3 and 6 to create a G-protein-binding pocket, as in Family A GPCRs. Pro321 in TM6 appears to act as a pivot. At the base of TMs 2 and 3, Arg151, His155 and Glu211 may form a loose equivalent of the Family A DRY (Asp-Arg-Tyr) motif. Although the details of this proposed activation mechanism clearly do not apply to all Family B GPCRs, the broad outlines may be conserved.

Download Full-text

Exploring Ligand Binding to Calcitonin Gene-Related Peptide Receptors

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.720561 ◽

2021 ◽

Vol 8 ◽

Author(s):

Giuseppe Deganutti ◽

Silvia Atanasio ◽

Roxana-Maria Rujan ◽

Patrick M. Sexton ◽

Denise Wootten ◽

...

Keyword(s):

Molecular Dynamics ◽

Ligand Binding ◽

Calcitonin Gene Related Peptide ◽

Related Peptide ◽

Calcitonin Gene ◽

Approved Drugs ◽

G Protein Coupled ◽

Dynamic Docking ◽

Receptor Family

Class B1 G protein-coupled receptors (GPCRs) are important targets for many diseases, including cancer, diabetes, and heart disease. All the approved drugs for this receptor family are peptides that mimic the endogenous activating hormones. An understanding of how agonists bind and activate class B1 GPCRs is fundamental for the development of therapeutic small molecules. We combined supervised molecular dynamics (SuMD) and classic molecular dynamics (cMD) simulations to study the binding of the calcitonin gene-related peptide (CGRP) to the CGRP receptor (CGRPR). We also evaluated the association and dissociation of the antagonist telcagepant from the extracellular domain (ECD) of CGRPR and the water network perturbation upon binding. This study, which represents the first example of dynamic docking of a class B1 GPCR peptide, delivers insights on several aspects of ligand binding to CGRPR, expanding understanding of the role of the ECD and the receptor-activity modifying protein 1 (RAMP1) on agonist selectivity.

Download Full-text

The Role of Cgrp-Receptor Component Protein (Rcp) in Cgrp-Mediated Signal Transduction

The Scientific World JOURNAL ◽

10.1100/tsw.2001.435 ◽

2001 ◽

Vol 1 ◽

pp. 12-12

Author(s):

M. A. Prado ◽

B. Evans-Bain ◽

S. L. Santi ◽

I. M. Dickerson

Keyword(s):

Signal Transduction ◽

Cgrp Receptor ◽

Nih3t3 Cells ◽

New Class ◽

Receptor Component ◽

Calcitonin Gene ◽

Cellular Signal ◽

Signal Transduction Proteins ◽

Component Protein

The calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

Download Full-text

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

International Journal of Molecular Sciences ◽

10.3390/ijms21113932 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3932 ◽

Cited By ~ 2

Author(s):

Preeti Kumari Chaudhary ◽

Sanggu Kim ◽

Youngheun Jee ◽

Seung-Hun Lee ◽

Kyung-Mee Park ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Platelet Activation ◽

Functional Role ◽

Thrombus Formation ◽

Receptor Activation ◽

G Protein Coupled ◽

Gpcr Desensitization ◽

Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Download Full-text