CGRP Signal Transduction
Our lab is studying the neuropeptide calcitonin gene-related peptide (CGRP), focusing on the role of CGRP in physiology and pathophysiology, and on the biochemical mechanism of signal transduction at the CGRP receptor. CGRP is a 37 aa peptide with potent vasodilator activity, that has been studied for its role in regulating vascular tone. It has also been shown to be important for migraine, regulating immune function, and perception of pain. Despite these important physiologic roles, therapeutic strategies targeting CGRP and its receptor have been limited. This delay has primarily been due to the fact that CGRP is a peptide, and thus has a complicated biosynthesis with a short half-life, and also due to the fact that the receptor for CGRP has turned out to be a complex of proteins, rather than a single molecule as predicted.
The complexity of the receptor for CGRP is particularly important. One of the central questions in biology today is how a cell converts an extracellular signal to an intracellular chemical signal that the cell can interpret. For example, extracellar hormones by themselves have no meaning to a cell until they bind their cognate cellular receptor and stimulate an intracellular signal.
This process, known as signal transduction, involves a change in the three-dimensional structure of the receptor, which results in activation of intracellular signaling mechanisms. Such intracellar signals are known as second messengers, and can take the form of chemicals produced in response to receptor activation such as cyclic adenosine monophosphate (cAMP), cGMP, inositol tris phosphate (IP3), or as altered ion concentrations (sodium, potassium, calcium, chloride) as a result of ion channel activation, or as activation of kinases or phosphatases, or changes in gene expression.
With such a wide array of intracellular responses, it is not surprising that the overwhelming majority of pharmaceutical targets today are receptors. Greater than 25% of all pharmacologics target one type of receptor: receptors that couple (signal) through GTP-binding proteins (G-proteins). G-protein coupled receptors (GPCRs) comprise the largest gene family known, and are shared between all eukaryotes, from mammals to yeast.
Ligands for GCPRs range from photons of light for the GCPR rhodopsin in the eye, to dimers of large (30 kDa) glycosylated protein hormones such as follicle stimulating hormone (FSH) in the gonads. GCPRs have a stereotypic structure which passes through the cell membrane 7-times, leading to the descriptor "hepta-helical", with an NH-terminus on the extracellular side of the membrane and the COOH-terminus on the intracellular side. GCPRs interact with an intracellular G-protein trimer, composed of an α,β, and γ subunit. Upon binding ligand, GCPRs undergo a conformational change that results in GTP incorporation into the α subunit of the G-protein trimer, which results in separation and activation of the α subunit and the βγ subunit pair. The G-protein subunits induce second messenger pathways until the GTP on the α subunit is hydrolyzed to GDP, at which time the subunits reform into the inactive αβγ trimer.
CGRP labeled with Alexa488 separated by reverse phase high performance chromatography (RP-HPLC). Red, unlabeled CGRP; Green, labeled CGRP peptides; Blue, mock labeling reaction.
It was once thought that G protein-coupled receptors (GPCRs) functioned as monomers in the cell membrane. However, recent data support the model that many GCPRs require homo- and heterodimerization for function. We discovered an intracellular protein required for activation of the GCPR for CGRP. The receptor for CGRP is an attractive target for development of therapeutics given the wide range of physiologic roles ascribed to CGRP. However, the CGRP receptor was difficult to identify, and cloning and characterization was the result of work in multiple labs. We now know that one of the reasons the CGRP receptor was so difficult to identify is that it is composed of three proteins:
- A ligand-binding receptor called calcitonin-like receptor (CLR) that has the stereotype GCPR heptahelical transmembrame conformation,
- An accessory protein named receptor activity modifying protein (RAMP1) that acts as a molecular chaperone and also contributes pharmacologic specificity, and
- A second accessory protein named receptor component protein (RCP) that couples the CLR/RAMP1 complex to the cellular signaling machinery.
The role of RAMP1 was discovered fairly quickly, but the role of RCP has been complicated.
Our laboratory is investigating the mechanism of RCP action by:
- molecular and biochemical requirements for RCP,
- proteins that interact with RCP in a functional receptor complex using proteomic strategies, and
- the role of RCP and CGRP in vivo using targeted homologous recombination.
- Fluoxetine modulates breast cancer metastasis to the brain in a murine model. BMC Cancer. 14, 598. (2014 Aug 16).
- Peripheral calcitonin gene-related Peptide receptor activation and mechanical sensitization of the joint in rat models of osteoarthritis pain. Arthritis Rheumatol. 66, 2188-200. (2014 Aug 01).
- Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis. J Neuroimmunol. 271, 18-29. (2014 Jun 15).