Mechanisms to terminate signalling

Cellular responses to agonists of the GPCRs are usually rapidly attenuated. Signals may be attenuated by mechanisms that operate at the level of the agonist, the receptor, the G proteins and at numerous downstream steps of the signalling pathway.

Several processes contribute to the removal of hormones and neurotransmitters from the extracellular fluid, but their relative importance depends on the nature of the agonist (Figure 130):

• For all agonists, dilution in the extracellular fluid and subsequent excretion reduces concentrations to levels that are too low to produce detectable responses in target cells.

Figure 130 Release, reuptake and degradation of the biogenic amines dopamine (DA), acetylcholine (Ach) and the neuropeptide substance (SP). Reproduced with permission, from Bohm, S. K., Grady, E. F. and Bunnett, N. W., 1997, Biochemical Journal, 322, 1-18. © The Biochemical Society.

• Uptake by high-affinity transporters is one of the most widespread mechanisms for removal of agonists of GPCRs and ligand-gated ion channels from the synapse. The importance of transporters in terminating neurotransmission has been well documented. Certain powerful psychoactive drugs exert their effects by inhibiting transporters and thereby prolonging the effects of the neurotransmitters: cocaine blocks the uptake of noradrenaline and dopamine.

• Extracellular degradation is the major mechanism for removing acetylcholine and peptides from the extracellular fluid. Acetylcholine released from nerve terminals is not taken up, but is degraded by acetylcholinesterase. Inhibition of this enzyme prolongs synaptic transmission, which ultimately desensitizes cholinergic receptors and results in paralysis and death due to respiratory failure. Indeed, nerve gases such as Sarin are acetylcholinesterase inhibitors. Peptides are removed from the extracellular fluid by enzymatic degradation, mainly by cell-surface peptidases.

Receptors are not static entities. The most extensively studied, and probably the most common form of regulation involves the reduction in responsiveness of a receptor upon prolonged stimulation by an agonist. This process, denominated as 'desensitization', 'tolerance', 'refractoriness' or 'tachyphylaxis', probably occurs as a measure to prevent cell damage:

• If an agonist only affects the activity of its own receptor, we may speak about 'homologous desensitization'. This form of desensitization takes place with P-arrestin binding since it only precludes coupling between the activated receptor and G proteins. This process affects no other receptor types (Figure 131).

• On the other hand, many examples are known where the activity of a receptor is either increased or decreased by unrelated drugs (e.g. drugs that interact with other receptors) and this form of regulation is termed 'heterologous desensitization'.

Figure 131 a) Homologous desensitization: only the agonist-occupied receptor is desensitized by a GRK/arrestin mechanism. b) Heterologous desensitization: activated PKA or PKC phospho-rylates and desensitizes different types of receptors. Reprinted from Trends in Pharmacological Science, 17, Chuang, T. T., Iacovelli, L., Sallese, M. and De Blasi, A., G protein-coupled receptors: heterologous regulation of homologous desensitization and its implications, 416-421. Copyright (1996), with permission from Elsevier.

Figure 131 a) Homologous desensitization: only the agonist-occupied receptor is desensitized by a GRK/arrestin mechanism. b) Heterologous desensitization: activated PKA or PKC phospho-rylates and desensitizes different types of receptors. Reprinted from Trends in Pharmacological Science, 17, Chuang, T. T., Iacovelli, L., Sallese, M. and De Blasi, A., G protein-coupled receptors: heterologous regulation of homologous desensitization and its implications, 416-421. Copyright (1996), with permission from Elsevier.

This form of desensitization takes place with second-messenger-dependent protein kinases, including cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) (Figure 131). These kinases are also able to phosphor-ylate serine and threonine residues within the cytoplasmic loops and C-terminal tail domains of many GPCRs. This phosphorylation process is sufficient to impair receptor-G protein coupling, but agonist occupancy of the target GPCR is not required. This implies that the activation of second-messenger-dependent protein kinases by one receptor is able to desensitize receptors for other ligands as well. In this respect, PKC activation leads to the phosphorylation and desensitization of many Gi- and Gq-linked GPCRs.

There are also additional differences between second-messenger kinase- and GRK-mediated GPCR phosphorylation:

• Whereas no consensus sites were found for phosphorylation by the GRKs, such sites clearly exist for the second-messenger kinases.

• Whereas p-arrestins display increased affinity for GRK-phosphorylated receptors, their affinity is unaffected by PKA- and PKC-induced phosphorylation. This is in line with the view that p-arrestin contributes to homologous, but not heterologous, desensitization.

• PKA and PKC are thought to represent the predominant mechanisms by which GPCR desensitization is achieved at low agonist concentrations whereas GRK/ p-arrestin- mediated receptor phosphorylation is thought to represent GPCR desensitization at high agonist concentrations. Indeed, because of the existence of a 'receptor reserve' in many cells, second-messenger kinase activity will be more pronounced than the receptor occupancy at low agonist concentrations. Concentrations of agonists with high efficiency giving only 1-2% receptor occupancy, may therefore be sufficient to fully stimulate PKA- and PKC-mediated phosphorylation in most cells. In contrast, the EC50 for GRK-mediated phosphorylation is much higher, approaching the KD for agonist binding. Thus, as the concentration of an agonist with high efficacy increases, GPCR desensitization gradually swings from second-messenger kinase-mediated to P-ARK-mediated.

Finally, GPCR signalling can also be terminated at the level of the G protein. For example, a family of proteins, termed regulators of G protein signalling (RGS) act to increase the rate of hydrolysis of GTP bound to both Gai and Gaq subunits, thereby dampening signalling via Gi- and Gq- regulated signalling pathways.

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