Kinetics of G‐protein‐coupled receptor signals in intact cells

Abstract

G-protein-coupled receptors (GPCRs) are the largest group of cell surface receptors. They are stimulated by a variety of stimuli and signal to different classes of effectors, including several types of ion channels and second messenger-generating enzymes. Recent technical advances, most importantly in the optical recording with energy transfer techniques--fluorescence and bioluminescence resonance energy transfer, FRET and BRET--, have permitted a detailed kinetic analysis of the individual steps of the signalling chain, ranging from ligand binding to the production of second messengers in intact cells. The transfer of information, which is initiated by ligand binding, triggers a signalling cascade that displays various rate-controlling steps at different levels. This review summarizes recent findings illustrating the speed and the complexity of this signalling system.

Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization

“…We chose mGluR1␤ because mGluR1's role in synaptic transmission has been extensively studied (16-18, 29, 30) and the ␤ subtype is much more efficiently addressed to the plasma membrane than the ␣ subtype, at least in the absence of specific scaffolding proteins (21,31). The activation rate we report (time course Յ 10 ms) is if the fastest reported so far for a ligand-gated GPCR (6)(7)(8)(9). This finding suggests that despite its remote extracellular location, the VFTM [which is homologous to the binding domain of ionotropic glutamate receptors that activate within tens of micro- seconds (15)] speeds up the overall conformational change induced by agonist binding.…”

“…These events are often delayed (by hundreds of milliseconds) and do not reflect the real-time activation of the receptor, that is, the conformational change from an inactive to an active state. Recently, activation of some class A (rhodopsin-like) GPCRs has been monitored in real-time by using a fluorescence resonance energy transfer (FRET)-based approach (6). The FRET responses observed upon agonist binding were well described by monoexponential fits whose time constants were faster than previously expected: approximately 40 ms for the ␣ 2A -adrenergic receptor (7), 70 ms for the A 2A -adenosine receptor (8), and 60 ms for the ␤ 1 -adrenergic receptor (9), although not as fast as for light receptor rhodopsin (10) and ligand-gated ion channels (11).…”

“…To investigate these kinetics, we used a FRET approach similar to that which led to the determination of the fast activation rate of members of the class A GPCRs (6)(7)(8)(9). Type 1 mGluRs function in synaptic transmission has attracted particular interest (16) since they were shown to mediate slow synaptic excitatory currents (17) and long-term plasticity (18).…”

Membrane heterogeneity – from lipid domains to curvature effects

“…Membrane heterogeneity is supposed to be involved in GPCR desensitization and internalization (13) and suspected to influence the kinetics of the signaling cascade (145). In the canonical model for GPCR signaling a ligand binds to the receptor on the outside of the cell, which, as a reaction, changes the conformation of its cytosolic part, see Fig.…”

G protein coupled growth factor receptor tyrosine kinase:no longer an oxymoron

“…Canonical signal transduction via trimeric G proteins is spatially and temporally restricted, i.e., triggered exclusively at the PM by agonist activation of GPCRs via a process that completes within a few hundred milliseconds. 6 Non-canonical transactivation of trimeric G proteins by RTKs via the GIV-CT platform has distinctive features. Such activation can be triggered by multiple growth factor RTKs as well as other receptors, 3 can occur at the PM and on internal membranes discontinuous with the PM 7,8 (Table 1), and can continue for prolonged periods of time (several minutes).…”