How do GPCRs activate G protein

G protein coupled receptors

In mammals, G protein-coupled receptors (GPCRs) are the most abundant superfamily of integral membrane proteins. They are important mediators for signal transmission, which interacts with signal molecules on the outer surface of the cell membrane and transmits the signal across the membrane. Examples of such signaling molecules are non-steroid hormones, neurotransmitters, odorous substances and (in the case of rhodopsin) even light. GPCRs can be found in all types of eukaryotes, from amoebas to fungi, plants, invertebrates, and vertebrates. The genome of the human body codes for almost 900 GPCRs, of which around 400 are olfactory receptors. The natural ligand has not been identified for many GPCRs; these receptors are called orphan GPCRs.

It has been estimated that ~ 50% of all drugs work by binding to GPCRs. Understanding signal transmission through GPCRs is therefore of great interest not only for basic research, but also for the pharmaceutical industry, as structural knowledge of GPCRs can be applied for drug design and virtual screening.

A typical GPCR consists of 7 membrane-spanning helices. These are connected by hydrophilic loops, which - depending on their occurrence - are called extracellular loops (EL1 - EL3) or intracellular loops (IL1 - IL3). They also have an extracellular N-terminal tail and an intracellular C-terminal tail, which in many cases is palmitoylated.

Signal transduction is initiated by binding a specific ligand to the receptor. Depending on the type of ligand, they bind to intramembrane binding sites (amines, nucleotides or lipids) or extracellular binding sites (proteinases, neuropeptides or proteohormones). There are two types of ligands for GPCRs: ligands that activate the receptor for signal transmission ("agonists"), and ligands that make the receptor temporarily inactive ("antagonists"). Interaction with an agonist changes the conformation of the GPCR, while interaction with an antagonist blocks the binding of other ligands and fixes the GPCR in an inactive conformation, thereby preventing any transmembrane signaling. There are also allosteric inhibitors.

The binding of an agonist induces a conformational change in the receptor, which is then transferred to its cytoplasmic side, where the GPCR interacts with heterotrimeric G proteins. These proteins (consisting of the subunits alpha, beta and gamma with a GDP / GTP binding site on their alpha subunit) only form a stable heterotrimer in their GDP form. It is still unclear whether heterotrimeric G proteins are constitutively bound to the GPCR or whether agonist-activated receptors randomly interact with the G proteins. The activated receptor now acts as a GDP / GTP exchange factor for the G protein bound to the GPCR on the cytoplasmic side of the membrane, causing the dissociation of the heterotrimeric G protein and the release of active GTP-Gα be induced. These Gα proteins now transduce the ligand-induced signal by activating enzymes that catalyze the production of second messengers (for example activation of adenylyl cyclase or phospholipase C). After a short time, both the intrinsic GTPase activity of Gα as well as other GTPase-activating proteins (GAPs) to hydrolyze the Gα-bound GTP, thereby causing the Gα becomes inactive and the formation of the heterotrimeric G protein (G.α / β / γ) is made possible).

As with most membrane proteins, however, all GPCRs (with the exception of rhodopsin, a light-activated GPCR) are only present in cells in very small amounts. Since milligrams of stable and homogeneous protein are a prerequisite for biophysical and structural investigations, we concentrate on the one hand on the heterologous production of recombinant GPCRs in various expression systems and on the other hand on the creation of purification protocols as well as biochemical and biophysical investigations and protein crystallization to determine the X-ray structure. In the past we have the baculovirus system and insect cells, Escherichia coli, various yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris) and the Semliki Forest virus-mediated expression in mammalian cell lines is used. In a genomic approach, we worked on more than 100 different GPCRs to identify the most suitable GPCRs. Right now we've reduced the number of GPCRs to fewer than a dozen. Depending on the receptor, these are in Pichia pastoris, expressed in insect or mammalian cells. We are also testing cell-free coupled transcription translation systems for the production of GPCRs.

Our aim is to determine the structure of native GPCRs and functional GPCR-protein complexes in their active state. Most of our targets come from mammalian species such as humans, mice, and rats. We also work with NMR groups (Prof. C. Glaubitz, University of Frankfurt, Prof. H. Oschkinat, FMP, Berlin) to determine the conformation of peptide ligands in the receptor-bound state using solid-state nuclear magnetic resonance spectroscopy.

Our structural studies are complemented by collaborative fluorescence spectroscopic techniques to obtain information about the oligomeric state of our GPCRs in their native membranes.