Arrestin in Pharmacology

Slide 1: They are well known negative regulators of G-protein-coupled receptor (GPCR) signalling. We are going to review their classical functions and highlight some of the new discoveries that have been found. As there has been recent evidence to suggest that in addition to their role as GPCR/G-protein signalling terminators they seem to have a role as adaptors and signal transducers from a variety of activated receptor types. (Touch on these later) Slide 2: This table summarises the arrestin proteins currently known. There are 4 arrestin proteins; Arrestin1 and 4 are confined to the retinal rods and cones and 2 and 3 (confusingly aka ?

-Arrestin 1 and 2) are ubiquitously expressed in mammalian tissues ? 1 is found to be located in both the cytosol and the nucleus ? 2 is found in the cytosol and is excluded from the nucleus as it has a nuclear export domain at the c-terminus. Slide 3: ?1 and ? 2 proteins are 78% identical in their AA sequence and structurally differ mainly in C termini. ?-arrestins contain two major domains: an N domain and a C domain; The N domain (1-185) is indispensable for the nuclear localisation of both arrestins. There is a Nuclear export signal which is located at the C-terminus of ? -arrestin 2.

The C-terminus allows the initiation of the clathrin-dependent endocytosis. One of the classical functions of arrestins. There are also IP6-binding site residues in the C domain at position 233-251 Functionally the ? -arrestins can often substitute each other as mice with genetic deletion of either isoform display normal function. However, a double ? 1 and 2 K/O is embryonically lethal. Slide 4: Desensitisation = the decreased signalling of GPCRs to repeated or sustained stimulation with ligand agonists. (An adaptive property of cells to prevent potentially harmful responses due to excessive receptor activation).

Ligand agonist binding to GPCRs results in recruitment of GRKs into close proximity to the agonist-bound receptor. The GRKs phosphorylate specific serine and threonine residues in their intracellular loops and c termini of the agonist-occupied receptors, thereby converting the receptors to high affinity binders to ? -Arrestins. 7 GRKs have been identified and cloned, with GRK2 and 3 being most extensively studied. Both GRK2 and 3 bind to free ?? subunits and thus are recruited to the plasma membrane following G-p activation by the GPCRs, this aids in the localisation upon receptor activation.

This inactivates the GPCR as it physically blocks its interaction with the G-protein. More recently, the ? arrestins were also shown to facilitate the dampening of receptor/G-protein signalling via increased breakdown of 2nd messengers such as cAMP and diacyglycerol. The other classical function of ? -Arrestins is to serve as adaptors which facilitate the clathrin mediated endocytosis of the receptors in response to agonists. This function requires the ubiquitination of ? -Arrestins which leads to the interaction of ? -arrestins with clathrin and the clathrin adaptor AP-2. Slide 5:

There has been new evidence to now suggest that ? -arrestins also facilitate the endocytosis of other GPCR subtypes such as the frizzled-4 receptor and non-GPCR such as TGF-B receptors and insulin growth factor receptors. Slide 6: In addition to the role in receptor desensitization and internalization. Recent evidence also suggested that ? -arrestin is involved in signaling transduction, which act as adaptors or scaffold to link GPCRs to signaling pathways such as the ERK. Slide 7: In order to show that ? -arrestins are involved in independent signalling, this simple experiment was conducted by Robert Lefkowitz.

When AT-2 receptors are activated they cause the activation of the Erk; this was measured. Cells were transfected to express the AT receptor; in one of the cells the AT-R was mutated. The cells were treated with different agonists; the first WT AT-R was treated with 160nM of AT, the second WT AT-R was treated with an SII agonist, the mutated AT receptor was treated with AT-2. The cells in our control gave results as you’d expect;

AT wild type agonist gives 100% activation as you would expect and the WT SII AT when applied to this cell also leads to Erk activation (approx.half of the wildtype AT11) and the same is true for the mutated AT In the last two there is no G-protein activation involved yet we are getting half as much Erk activation as in the WT Now the same experiment was done with siRNA (small interfering) to silence the ?

-arrestins expression and the recorded activation of Merk for the WT Ang 11 drops by half but we completely remove Erk activation from the WT SII and the mutated receptor. This suggests that they were activating the ERK system exclusively via ? -arrestins in the control experiment.

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