Agonist-selective mechanisms of GPCR desensitization

Abstract

The widely accepted model of G protein-coupled receptor (GPCR) regulation describes a system where the agonist-activated receptors couple to G proteins to induce a cellular response, and are subsequently phosphorylated by a family of kinases called the G protein-coupled receptor kinases (GRKs). The GRK-phosphorylated receptor then acts as a substrate for the binding of a family of proteins called arrestins, which uncouple the receptor and G protein so desensitizing the agonist-induced response. Other kinases, principally the second messenger-dependent protein kinases, are also known to play a role in the desensitization of many GPCR responses. It is now clear that there are subtle and complex interactions between GRKs and second messenger-dependent protein kinases in the regulation of GPCR function. Functional selectivity describes the ability of agonists to stabilize different active conformations of the same GPCR. With regard to desensitization, distinct agonist-activated conformations of a GPCR could undergo different molecular mechanisms of desensitization. An example of this is the mu opioid receptor (MOPr), where the agonists morphine and [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) induce desensitization of the MOPr by different mechanisms, largely protein kinase C (PKC)- or GRK-dependent, respectively. This can be best explained by supposing that these two agonists stabilize distinct conformations of the MOPr, which are nevertheless able to couple to the relevant G-proteins and produce similar responses, yet are sufficiently different to trigger different regulatory processes. There is evidence that other GPCRs also undergo agonist-selective desensitization, but the full therapeutic consequences of this phenomenon await further detailed study.

Figure 1

G protein-coupled receptor (GPCR) regulation by G protein-coupled receptor kinases (GRKs) and second messenger-dependent protein kinases. (a) The classical model of GPCR regulation by GRKs and arrestins. The GPCR is activated by agonist (1) leading to G protein coupling (2) and effector modulation. The agonist-occupied GPCR is subsequently phosphorylated by GRK (3), and arrestin binds to the phosphorylated GPCR, leading to receptor desensitization (4), internalization (5), dephosphorylation (6) and recycling (7) of the GPCR. Particularly with longer agonist treatments, internalized GPCR may also be targeted for downregulation. (b) GPCR regulation by second messenger-dependent protein kinases, in this example protein kinase A (PKA). Here, the agonist binds to the GPCR (1) leading to G_s activation (2) and increased levels of cyclic AMP, which activates PKA. The kinase is then able to phosphorylate both agonist occupied (3) and unoccupied (4) GPCRs. This phosphorylation causes desensitization by uncoupling the GPCR from G protein or in the case of the agonist unoccupied receptor by preventing GPCR coupling to G protein. Whether or not GPCR phosphorylation by second messenger-dependent protein kinases leads to arrestin binding or internalization depends upon the particular GPCR subtype in question. (c) Direct and indirect mechanisms of GPCR regulation by second messenger-dependent protein kinases. In this example, activated protein kinase C (PKC) can directly phosphorylate and desensitize the GPCR (1), or PKC can phosphorylate and activate GRK2, which consequently has an enhanced ability to phosphorylate the GPCR (2), or PKC can phosphorylate other, as yet unidentified, regulatory proteins, which then effect GPCR desensitization (3).

Figure 2

Functional selectivity of agonist action at G protein-coupled receptors (GPCRs). (a) The traditional ‘linear' model of agonist action at a GPCR. In this case, the red and yellow agonists, as well as any other agonist at this GPCR, produce the same set of cellular responses as they each stabilize the same active conformation of the receptor, which couples to the same set of G proteins. (b) Functional selectivity: the red and yellow agonists each stabilizes a different active conformation of the GPCR, which couples to a different array of G proteins. These two agonists therefore produce different cellular response profiles in this tissue. It is interesting to note, however, that functional selectivity would be missed if only the response to the G protein illustrated in brown was measured in a screen for agonist activity.

Figure 3

Models of functional selectivity in the agonist-induced desensitization of μ-opioid receptor (MOPr). (a) Depicts the sequential activation of the receptor, with morphine being able to induce only the MOPr^* state of the receptor, whereas DAMGO is capable of inducing MOPr^* and then MOPr^**. The MOPr^* conformation undergoes protein kinase C (PKC)-mediated desensitization, whereas the MOPr^** conformation undergoes G protein-coupled receptor kinase (GRK)/arrestin-mediated desensitization. In (b), morphine and DAMGO directly stabilize different active conformations of MOPr, MOPr^* and MOPr^**, respectively. MOPr^* and MOPr^** both couple to G protein-coupled inwardly rectifying K⁺ current, but undergo different mechanisms of desensitization. It is also possible that MOPr^* and MOPr^** regulate other signalling pathways differentially, such as arrestin-dependent MAP kinase activity.

Figure 4

Diagrammatic representation of the possible effects of changes in the cellular concentrations of regulatory proteins on the mechanism of μ-opioid receptor (MOPr) desensitization. In (a), morphine desensitization of MOPr is mediated principally by protein kinase C (PKC), whereas desensitization of MOPr by agonists such as Met-Enkephalin and DAMGO is mediated predominantly by G protein-coupled receptor kinases (GRKs). (b) In a neuron with high levels of GRKs or in an experimental cell system where GRKs have been overexpressed, GRK-mediated desensitization may now play a significant role in morphine-mediated MOPr desensitization; however, Met-Enkephalin- and DAMGO-mediated desensitization will still involve predominantly GRKs.