Question

Discuss in detail how Trimeric G-protein signaling pathways would be influenced by the following:

- Please describe the molecular switches that are applied in G-protein coupled receptor signaling pathways, and compare the molecular switches in detail

Answer 1

Trimeric G- protein

Trimeric G Proteins are usually found coupled to a receptor protein and attached to the cytoplasmic face of the plasma membrane. It's function is to couple the receptor molecule to either an ion channel or enzyme (target signal Protein) where it acts as a relay Protein . Trimeric G Proteins are composed of three sub-units, alpha beta and gamma. In its inactive state the alpha sub-unit is bound to GDP, when the coupled receptor is activated this alpha sub-unit releases this GDP molecule. A GTP molecule takes the place of the GDP on the alpha sub-unit and the protein under goes a large conformational change; the G protein is now in it's activated state. The sub-units dissociate into an alpha sub-unit and a beta-gamma complex which then go on to activate their target molecules which may be enzymes, this therefore carries the signal forward and the result is a cell response .

Guanosine triphosphate (GTP)-binding proteins (G-proteins) are the regulatory GTPases that have the ability to bind GTP and hydrolyze it to guanosine diphosphate (GDP). GDP locks G proteins into their inactive state, while GTP locks G-proteins into their activated state. Active or inactive states of G-proteins depend on the binding of GTP or GDP, respectively. G-proteins have been found to be key players in plant innate immunity. The GTPases act as molecular switches controlling the transmission of extracellular signals like pathogen-associated molecular patterns (PAMPs) to intracellular signaling pathways. The PAMPs have been shown to activate GTP binding to G-protein. The GTPase is normally inactive. The PAMP stimulates exchange of GTP for GDP and thus converts the G-proteins from their inactive state to their active state. Upon stimulation by an upstream PAMP signal, a guanine nucleotide exchange factor (GEF) converts the GDP-bound inactive form into the GTP-bound active form through GDP/GTP replacement. Through its effector domain, the GTP form interacts with specific downstream effector proteins. The GTP form exhibits a weak intrinsic GTPase activity for GTP hydrolysis, requiring a GTPase-activating protein (GAP) for efficient deactivation. Most small GTPases cycle between membrane-bound and cytosolic forms. Only membrane-associated GTPases can be activated by GEF and their removal by a cytosolic factor called guanine nucleotide dissociation inhibitor (GDI) negatively regulates these GTPases.

Action of molecular switches in G protein Coupled reactors(GPCRs)

Molecular Switches that are applied in G-protein coupled receptor signaling pathway, G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called "molecular switches" buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homoand heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs.

Molecular Switches

Signalling protein can act as molecular switches. Allosteric regulation by binding small molecules is a widespread regulatory mechanism for the activity of many proteins, including receptors and structural, motor and signalling proteins, the addition or loss of phosphate groups usually drives most functional changes in the sequence of activation/deactivation steps that form a typical intracellular signalling pathway. In reality, many intracellular signalling proteins act as molecular switches. What often happens is that the proteins can be temporarily modified, converting them from an inactive (non-signalling) form to an active (signalling) form (Figure 2), or vice versa. Usually the upstream signal induces a change in the protein's conformation, which enables it to carry out its downstream signalling function. The reason why such molecules are sometimes referred to as molecular switches is because they are either ‘on’ or ‘off’. These proteins can be grouped according to how they are switched on/off, rather than their subsequent mode of action.