CGRP Receptor activation
The calcitonin gene-related peptide (CGRP) receptor is a complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). This receptor plays an important role in vasodilation, neurogenic inflammation and is involved in the pathology of migraine. Though mechanisms have been proposed, it is largely unknown how exactly the receptor is activated by its ligand CGRP. Recently a Cryo-EM structure of the human CGRP receptor in complex with CGRP was released, as well as data that displays the key residues within the receptor that are integral for receptor activation. It is known that CGRP forms functionally important interactions with the CLR–RAMP1 complex and so the aim for this project is to review current knowledge on CGRP receptor activation and to utilise data alongside molecular visualisation programs including VMD and SwissPdbViewer to analyse the potential mechanism by which CGRP activates its receptor.
Introduction to CGRP
CGRP is a 37 amino acid neuropeptide with two major isoforms (α and β) which belong to the calcitonin family of peptides. In humans, the two forms differ by three amino acids and so have very similar effects due to their amino acid sequence being alike (Ghatta and Nimmagadda., 2004). The receptor for CGRP is a secretin family receptor GPCR (Family B) known as CLR (Barwell et al., 2013). G-protein coupled receptors (GPCRs) receive a great deal of scientific interest due to being one of the largest targets for pharmaceuticals, however the CGRP receptor is unlike a typical GPCR as there is a requirement for the accessory protein RAMP1, a transmembrane protein which, when dimerised with CLR allows cell surface expression and CGRP binding to occur (Barwell et al., 2013). When alone, CLR will not reach the cell surface and will not respond to any known ligands (Hay et al., 2017).
Its been established that CGRP is a highly potent vasodilator and has a definite role in migraine but there is evidence CGRP has a range of actions from cardioprotective effects to pain and itch in arthritis, however, the pathophysiological states that CGRP is involved in is yet to be fully explored (Kee et al., 2018; Russell et al., 2014). As the CGRP pathway appears to be involved in many diseases and disorders, it is important to understand the activation mechanism to gain further knowledge of the CGRP receptor which in turn will lead to the design of emerging medicines.
The CGRP Receptor
The CGRP receptor consists of an extracellular domain and a transmembrane domain (TM). The extracellular domain is found outside the cell and is connected to the TM found within the cell membrane. CGRP causes the TM to change its conformation by interacting with the two domains. The C‐terminus of the peptide interacts with the N-terminal domain of CLR while the N‐terminus of the peptide interacts with the transmembrane domain of CLR and its associated extracellular loops resulting in activation (Woolley et al., 2013). The details of how CGRP interacts with the TM and extracellular loop regions of the CGRP receptor remains unclear (Barwell et al., 2012)
CGRP Binding and Understanding Receptor Activation
As of now, a structure of CGRP bound to its receptor has been proposed (Liang et al., 2018). Current literature suggests that 61.5% of CGRP’s surface is buried within the CLR-RAMP1 complex which indicates numerous significant points of contact between the bound peptide-receptor complex (Liang et al., 2018). The second extracellular loop (ECL2) is thought to have an important role in ligand binding and receptor activation as seen in other GPCRs (Woolley et al., 2013). 13 ECL2 residues were identified as influencing CGRP-receptor interaction. ECL2 is highly conserved and the longest ECL so there is certainly it has involvement in the activation of GPCRs (Woolley et l., 2013). ECL 1 has two residues involved in peptide binding and ECL3 has one, which suggests their roles have less influence over CGRP-receptor interactions although they are mandatory for normal functioning of the receptor (Barwell et al., 2011). The only direct contact between CGRP and RAMP1 is limited to the far C terminus of the peptide and a number of RAMP residues (Liang et al., 2018). The N-terminal peptide loop within CGRP forms extensive van der Waals interactions with CLR (Liang et al., 2018). There is also a functionally important interaction between the N terminus of CGRP and the core of CLR involving a hydrogen bond between H295 and CGRP T6. Other hydrogen bonds are present but in little number and are of little importance for CGRP binding (Liang et al., 2018). Below H295 is a peptide binding pocket formed by a series of amino acids, this pocket is of particular interest as it makes for an attractive target for new drugs (Liang et al., 2018). Other important interactions include those between the peptide and TM3/TM5 (Liang et al., 2018). Although the interactions between CGRP and its receptor are well known. It is uncertain how these interactions result in activation of the CGRP receptor.
Activation of the CGRP receptor has been associated with an increase in the second messenger cAMP (King and Brain., 2013). There have been a number of mutations and alanine substitutions to residues that have lead to a decrease in CGRP potency on cAMP accumulation, this would indicate that the affected residues have functional importance in receptor activation. Prior research has discovered that CGRP T4, CGRP T6, CGRP T9 among others are important residues for receptor activation (Liang et al., 2018).
Recent developments in cryo-electron microscopy have led to the structure of an active CGRP receptor. Figure 1 (Liang et al., 2018). Cryo-EM is a very powerful tool, in this case, it allows researchers to observe the CGRP receptor as a whole and in its active state as opposed to studying individual components of the receptor structure (Diamond Light Source., 2015). The cryo-EM model is now available on the Protein Data Bank (a database for the structural data of proteins and other large biological molecules) and will be used to gain an understanding of CGRP-receptor activation.
Figure 1, Complex of a full-length, active-state calcitonin gene-related peptide receptor with calcitonin gene-related peptide ligand (Liang et al., 2018)
The Need for New CGRP Agonists and Antagonists
Migraine medication has been developed in the form of CGRP receptor antagonists, all of which have shown promising results. The gepant class of drugs which are non-peptide CGRP antagonists, including olcegepant and telcagepant, have high selectivity for CGRP receptors and have shown therapeutic efficacy in migraine induced by CGRP. Despite this, the marketing of these drugs has been halted due to safety and tolerability issues including liver toxicity and poor oral bioavailability (Goadsby, P., 2016). CGRP receptor antibodies could be an appropriate alternative as they have demonstrated similar efficacy and good tolerability without inducing liver toxicity, but the long-term effects of blocking CGRP are unknown and the cost of therapy is an additional problem (Deen et al,. 2017). In recent news, Erunumab, a monoclonal antibody of the CGRP receptor, was licensed for use by the FDA and EMA. It is currently being evaluated by NICE for the preventative treatment of migraine so there is promise that CGRP antagonists could have a place in clinical practice in the UK (NICE., 2018). .
There is also a chance for CGRP receptor agonists to have a role in future healthcare. CGRP is a potent vasodilator and as a consequence, its deemed to have cardioprotective effects (Kee et al., 2018). Evidence shows that the peptide helps relieve congestive heart failure, but the degree of benefit is limited because CGRP has a relatively short half-life, and as a peptide, it cannot be delivered orally (Gennari et al., 1990; Shekhar et al., 1991). Analogues of the peptide have been synthesised which improved on the half-life and the beneficial effects in heart failure were demonstrated at a similar potency to the native peptide (Aubdool et al., 2017). To obtain an oral agonist, however, research most probably needs to be done in the area of non-peptide CGRP agonist synthesis. Further work here may uncover new drugs that could be viable for the treatment of cardiovascular disease.
Under these circumstances, there is encouragement for further research into the mechanism of CGRP receptor activation to develop drugs that could possibly be advantageous over existing treatments.
Aim and Objectives
Aim: The aim of this project is to analyse the mechanism by which CGRP activates its receptor, through the use of molecular visualisation programs VMD and SwissPdbViewer
– Become familiar with VMD and SwissPdbViewer
– Identify and record the residues that move during the transition from the inactive to the active state of the receptor
– Identify key points of contact between CGRP and the CLR-RAMP1 complex that have importance in receptor activation
Research Design and Methodology
In this project, the plan is to continue studying how CGRP binds to its receptor and how this causes the receptor to become activated. Learning how the receptor shape will change when CGRP binds to its receptor is achievable via computer modelling. The active-state receptor structure is known for certain, but by using homology modelling, we can make a good guess at the inactive structure.
This will be done using molecular dynamics simulations (VMD) and macromolecular modelling (SwissPdbViewer). Molecular dynamics (MD) is a form of computer simulation in which atoms and molecules are allowed to interact for a period of time giving a view of the motion of the atoms. It solves Newton’s equation of motion on a model of a molecule to obtain the trajectory of its motion (Jefferies et al., 2017). Using MD, Dr John Simms has built a model on how the CGRP receptor moves between the two states and by using VMD to analyse this trajectory, we can see what residues are moving frame by frame. A table will be constructed to record what is moving and at what frame this movement occurs. Conformations will also be saved at specific frames and viewed in SwissPdbViewer for analysis.
SwissPdbViewer allows viewing of several models simultaneously so the model receptor can be superimposed on the cryo-EM structure allowing for comparisons and as a form of quality control. It can also be used to obtain bond lengths, atom distances and find hydrogen bonds between proteins and ligands (Rhodes, G., 2006). Through this method, the interactions between CGRP and its receptor can be better understood which will add onto the existing body of academic research and potentially be beneficial in the design of new drugs.
- Aubdool, A. A., Thakore, P., Argunhan, F., Smillie, S.-J., Schnelle, M., Srivastava, S., et al. (2017). A novel α-calcitonin gene-related peptide analogue protects against end-organ damage in experimental hypertension, cardiac hypertrophy, and heart failure. Circulation, 136,pp. 367–383.
- Evaluation of the therapeutic potential of a CGRP analogue with a longer half-life than the native peptide. Testing its ability to relieve cardiovascular disease in murine models of hypertension and heart failure. Provides details of results in terms of cardioprotection and tolerability.
- Barwell, J., Conner, A. and Poyner, D. (2011). Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research, 1813(10), pp.1906-1916.
- Detailed analysis of the function of ECL1 and ECL3 recognising their importance in binding and cell surface expression. Provides information on the residues in the ECLs that are of importance in receptor expression. Provides some information on the interactions between CGRP and the domains of its receptor
- Barwell, J., Gingell, J., Watkins, H., Archbold, J., Poyner, D. and Hay, D. (2012). Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?. British Journal of Pharmacology, 166(1), pp.51-65.
- Reviews how applicable Family B GPCRs are to CTR and CLR receptors. This includes looking into the functional domains, N-terminal domains, C-terminal domains and transmembrane domains of the receptors.
- Barwell, J., Wheatley, M., Conner, AC., Taddese, B., Vohra, S., Reynolds, CA. and Poyner, DR. (2013), The activation of the CGRP receptor. Biochemical Society Transactions, vol. 41, no. 1, pp. 180-184.
- Talks about the activation of Family B GPCRs and how it could be applied to the CGRP receptor. Provides detailed background information on the CGRP receptor.
- Deen, M., Correnti, E., Kamm, K., Kelderman, T., Papetti, L., Rubio-Beltrán, E., Vigneri, S., Edvinsson, L. and Maassen Van Den Brink, A. (2017). Blocking CGRP in migraine patients – a review of pros and cons. The Journal of Headache and Pain, 18(1).
- A review of the advantages and disadvantages of blocking GCRP in migraine, looking at efficacy and tolerability of drugs including gepants and monoclonal antibodies. Concluding that the benefits of CGRP treatment of migraine outweigh the drawbacks. Provides information on the use of CGRP receptor antibodies for the preventative treatment of migraine.
- Diamond Light Source. (2015). How it Works: Cryo-Electron Microscopy – Diamond Light Source. [online] Diamond.ac.uk. Available at: https://www.diamond.ac.uk/Home/News/LatestFeatures/Issue-5/16_11_15.html [Accessed 1 Nov. 2018].
- A website which includes a guide to how cryo-electron microscopy works. Has information on how it is different from other forms of microscopy and in some scenarios, why it is a more appropriate technique.
- Gennari, C., Nami, R., Agnusdei, D., and Fischer, J. A. (1990). Improved cardiac performance with human calcitonin gene related peptide in patients with congestive heart failure. Cardiovasc. Res. 24,pp. 239–241.
- Looks at what cardiovascular effects CGRP has when administered to patients who have congestive heart failure. Provides results on the outcome of using CGRP treatment, as well as issues that would limit its use in a clinical setting.
- Ghatta S., Nimmagadda D. (2004) Calcitonin gene-related peptide: Understanding its role. Indian Journal of Pharmacology 36: 277-283.
- A review of the CGRP receptor and its biological significance in different systems of the human body. Includes useful introductory information on the CGRP receptor.
- Hay, D., Garelja, M., Poyner, D. and Walker, C. (2017). Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25. British Journal of Pharmacology, 175(1), pp.3-17.
- A review which provides details on the pharmacology on the CGRP family of peptides. Includes information to do with the importance of RAMP1 and what events would not occur without it.
- Jefferies, D. and Khalid, S. (2018). Modeling of Microscale Transport in Biological Processes. Academic Press, pp.1-18.
- Talks about recent advances in theoretical and computational modelling. Contains an introductory section about molecular dynamics in great detail.
- Kee, Z., Kodji, X. and Brain, S. (2018). The Role of Calcitonin Gene Related Peptide (CGRP) in Neurogenic Vasodilation and Its Cardioprotective Effects. Frontiers in Physiology, 9.
- A review discussing the potential of CGRP as a target in the treatment of cardiovascular diseases. Looking at CGRP antagonists, antibodies and analogues. Has information on the effects that CGRP exhibits including vasodilation, cardioprotection and involvement in migraine.
- King, R. and Brain, S. (2013). Handbook of Biologically Active Peptides. 2nd ed. San Diego: Academic Press, pp.1394-1401.
- Discusses all features of biologically active peptides including CGRP. Provides information in relation to CGRP and second messengers in terms of receptor activation.
- Liang, Y., Khoshouei, M., Deganutti, G., Glukhova, A., Koole, C., Peat, T., Radjainia, M., Plitzko, J., Baumeister, W., Miller, L., Hay, D., Christopoulos, A., Reynolds, C., Wootten, D. and Sexton, P. (2018). Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor. Nature, 561(7724), pp.492-497.
- A report of the cryo-EM active-state CGRP receptor structure. Provides detailed information on the interactions between CGRP and the binding site.
- Liang, Y., Khoshouei, M., Deganutti, G., Glukhova, A., Koole, C., Peat, T., Radjainia, M., Plitzko, J., Baumeister, W., Miller, L., Hay, D., Christopoulos, A., Reynolds, C., Wootten, D. and Sexton, P. (2018). 6E3Y Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor. [image] Available at: http://www.rcsb.org/structure/6E3Y [Accessed 10 Nov. 2018].
- Image source for the cryo-EM of the active human CGRP receptor.
- NICE (2018). Erenumab for preventing migraine [ID1188] | Guidance and guidelines | NICE. [online] Nice.org.uk. Available at: https://www.nice.org.uk/guidance/indevelopment/gid-ta10302 [Accessed 10 Nov. 2018].
- NICE evaluation of erenumab for prevention of migraine. It is ongoing and has a timeline of the events that have occurred thus far.
- Rhodes, G. (2006). Crystallography Made Crystal Clear. 3rd ed. Saint Louis: Elsevier Science.
- A guide for users of macromolecular models. Provides an introduction to SwissPdbViewer and a tutorial for beginners.
- Russell, F., King, R., Smillie, S., Kodji, X. and Brain, S. (2014). Calcitonin Gene-Related Peptide: Physiology and Pathophysiology. Physiological Reviews, 94(4), pp.1099-1142.
- A review of the physiology of CGRP including its synthesis, metabolism, structure, distribution as well as the CGRP receptor. Provides information on the pathophysiological states that CGRP is involved in.
- Shekhar, Y. C., Anand, I. S., Sarma, R., Ferrari, R., Wahi, P. L., and Poole-Wilson, P. A. (1991). Effects of prolonged infusion of human alpha calcitonin gene-related peptide on hemodynamics, renal blood flow and hormone levels in congestive heart failure. Am. J. Cardiol.67,pp. 732–736.
- A study looking at the hemodynamic effects of infusion of CGRP over a long period of time. Mentions limitations to treatment including loss of effect within 30 minutes after treatment was stopped.
- Woolley, M., Watkins, H., Taddese, B., Karakullukcu, Z., Barwell, J., Smith, K., Hay, D., Poyner, D., Reynolds, C. and Conner, A. (2013). The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach. Journal of The Royal Society Interface, 10(88), pp.20130589-20130589.
- Investigation of the role of ECL2 in the binding of CGRP to its receptor through the use of mutagenesis and modelling. Has detailed information on ECL2 and its involvement in CGRP receptor-ligand binding.