BBSRC Addressing the architecture, dynamics and activation mechanism of the CGRP
receptor.
Lay Summary
G protein coupled receptors (GPCRs) are the largest family of proteins in the human genome and
also the largest target for therapeutic drugs; thus they are of enormous scientific and practical
interest. They are divided into a number of families. Of these, family-A is the best understood, but
family-B includes receptors which are likely to be important in many disease states and so it is
important to understand how these function, both to further our knowledge of fundamental biology
and also for the design of new drugs.
Calcitonin gene-related peptide (CGRP) is found throughout the nervous system and is particularly
important in regulating both the cardiovascular system (the heart and blood vessels) and also the
immune system and inflammation. The receptor for CGRP is of special scientific interest as it involves
a GPCR called CLR and also a second protein called RAMP1. RAMP1 is a member of a protein family
that modulates a number of GPCRs of which the best characterised is CLR. CGRP is also likely to be
important both in cardiovascular disorders and any disease that involves inflammation. The peptide
is a major cause of migraine and drugs which block CGRP receptors have shown great promise in
clinical trials; however, so far it has not been possible to use these clinically because of toxicity
problems. Thus, there is an urgent need to develop new drugs that could act on CGRP receptors.
The CGRP receptor is made up of two parts. A portion called the transmembrane domain is found in
the membranes of cells. This is connected to the extracellular domain, which is on the outside of
cells. CGRP interacts with both parts of this structure and causes the transmembrane domain to
change shape. This causes the receptor to interact with other proteins, leading to cell activation. We
have a crystal structure of the part of the CGRP receptor that is on the outside of cells.
Unfortunately, we do not know how CGRP binds to this, nor do we know how it binds to the
transmembrane domain. This severely limits our understanding of the receptor and our ability to
design drugs that could target it.
We have previously used experimental data from a technique known as site-directed mutagenesis to
construct a computer model of the transmembrane domain of the CGRP receptor. This
transmembrane domain is very similar to the transmembrane domains of two family-B GPCRs which
were crystallised after our computer model was produced. This gives us confidence that our
approach of combining experimental and computational methods is valuable. In this project, we
intend to extend the approach to study how CGRP binds to both domains of the receptor and how
this causes the receptor to become activated. We will use mutagenesis and also methods where we
physically cross-link CGRP to the receptor to identify contact points. We will then use these to
construct computational models, which we can refine through further experimentation. Using a
computer, we can predict how the receptor shape will change when CGRP binds to it, so identifying
the mechanism for receptor activation. This knowledge will be benefitial in the design of new drugs
which can either block the receptor or promote its activation.
Technical Summary
The CGRP receptor is a particularly interesting family B G-protein coupled receptor (GPCR) having an
absolute requirement for an auxiliary protein known as Receptor activity modifying protein 1
(RAMP1). Class B GPCRs consist of a large extracellular domain (ECD) and a transmembrane domain
(TMD). They frequently associate with accessory proteins belonging to the family of RAMPs. They act
as receptors for a number of peptide hormones and neurotransmitters. They are attractive
therapeutic targets but it has proved very difficult to obtain drugs that target them. Several crystal
structures exist for the ECDs and there are crystal structures for two class B GPCRs (glucagon and
CRF), but neither have bound peptides and the orientation between the TMD and ECD for any
receptor remains speculative, as does the mechanism whereby agonists activate the receptors.
We have recently used a combination of site-directed mutagenesis and molecular modelling to
propose a structure for CGRP bound to the TMD of CLR. This shows excellent agreement with the
crystal structures, which were published after our modelled structures were deposited. Thus we
propose that our methodology is robust. Furthermore, the presence of the RAMP provides
additional constraints on the orientation of the ECD relative to the TMD, making the CGRP receptor
especially amenable to modelling by greatly reducing the number of ways in which it could be
modelled incorrectly.
We propose a strategy of photoaffinity cross-linking, disulphide trapping and point mutagenesis to
provide experimental information on the architecture of the receptor when bound to CGRP and as a
test for the modelling. This information will then be used to produce a model of the complex. We
will use molecular dynamics and other modelling techniques to plan the experiments, to interpret
the results and hence to determine the conformational changes caused by CGRP binding and so
establish how the receptor is activated by its native agonist.
Progress
We have now started to identify key contacts
between CGRP and its receptor, using the photoaffinity cross-linking. This has needed some
optimisation in order to get it to work on membrane
proteins. The picture shows our latest model for how
CGRP binds to its receptor. The transmembrane
bundle of CLR is white, the N-terminus of CGRP is in
green and the C-terminus of Gs (the G protein that
stimulates cAMP production and is needed for high
affinity CGRP binding) is in blue. CGRP contacts the
extracellular loops but also penetrates into the upper
third of the transmembrane bundle. This allows it to
contact amino acids on CLR which change the
orientation of the transmembrane bundle and allow
binding of Gs.
We are continuing to map contacts between the receptor and CGRP and to explore how these result
in receptor activation. The binding pocket we are uncovering is an attractive target for new drugs.