G-protein coupled receptor kinases as modulators of G-protein signalling

Abstract

G-protein coupled receptors (GPCRs) comprise one of the largest classes of signalling molecules. A wide diversity of activating ligands induce the active conformation of GPCRs and lead to signalling via heterotrimeric G-proteins and downstream effectors. In addition, a complex series of reactions participate in the 'turn-off' of GPCRs in both physiological and pharmacological settings. Some key players in the inactivation or 'desensitization' of GPCRs have been identified, whereas others remain the target of ongoing studies. G-protein coupled receptor kinases (GRKs) specifically phosphorylate activated GPCRs and initiate homologous desensitization. Uncoupling proteins, such as members of the arrestin family, bind to the phosphorylated and activated GPCRs and cause desensitization by precluding further interactions of the GPCRs and G-proteins. Adaptor proteins, including arrestins, and endocytic machinery participate in the internalization of GPCRs away from their normal signalling milieu. In this review we discuss the roles of these regulatory molecules as modulators of GPCR signalling.

Figure 1. Schematic model of G-protein signalling cycle

In the non-activated state, the GPCR is in the inactive conformation, and GDP is bound to the heterotrimeric G-protein. Upon agonist binding, the GPCR undergoes a conformational change to the activated state and is able to bind to the heterotrimeric G-proteins and act as a guanine nucleotide exchange factor. This causes the release of GDP and allows GTP to bind to the G-protein α-subunit. Following the nucleotide exchange the G-protein can dissociate into the GTP bound α-subunits and the βγ-dimer. Both the α-subunit and the βγ-dimer can interact with effector molecules such as ion channels (E1) or membrane bound enzymes (E2) and modulate their activity. The deactivation of signalling is initiated by the hydrolysis of GTP by the α-subunit. This reaction can be accelerated by proteins termed regulators of G-protein signalling (RGS) which have been shown to directly bind to the α-subunit of G-proteins. In the GDP bound state the α-subunit reassembles with the βγ-dimer to form the inactive heterotrimer.

Figure 2. Scheme of GRK-dependent modulation of GPCR-mediated signalling

After activation of GPCRs by ligands (L), the receptors activate heterotrimeric G-proteins as described in Fig. 1. In the case of GRK2/3, the βγ-subunits of G-proteins may recruit GRKs to the membrane to allow for GPCR phosphorylation (P) of activated GPCRs. Phosphorylation by GRKs allows the GPCRs to interact with uncoupling proteins such as arrestins which cause uncoupling of the GPCRs from G-proteins. In addition to the uncoupling reaction, many GPCRs undergo internalization. This can be achieved by targeting of the phosphorylated receptor by an adaptor protein to endocytotic pathways. Internalization of the β₂AR proceeds subsequent to the R-G-uncoupling step; the adaptor is a non-visual arrestin that targets the β₂AR to clathrin-coated pits. For some GPCRs, internalization may proceed in a manner that is independent of the uncoupling reaction and may involve yet to be defined adaptors and endocytic machinery. After endocytosis, the GPCRs may undergo lysosomal degradation or recycle to the surface membrane. Eff, effector molecule.

Figure 3. Multiple pathways of endocytosis for GPCRs

A, after agonist binding and activation of GPCRs, many GPCRs may undergo phosphorylation (P) by GRKs or other protein kinases. Phosphorylation can serve as a signal for the subsequent binding of certain adaptor proteins, but other mechanisms might also be operative, and might involve phosphorylation-independent binding to adaptors or direct association of the GPCRs with endocytic machinery. Subsequently, the GPCRs can be directed to different endocytic pathways. B, certain pathways are dynamin (D) dependent including internalization pathways that use arrestin as the adaptor and clathrin-coated pits for endocytosis as has been demonstrated for β₂-adrenergic (β₂AR) and μ-opioid receptors (MOR) (Zhang et al. 1997; Whistler & von Zastrow, 1998). Localization and/or internalization of GPCRs via caveolae may also occur and require dynamin for fission (Oh et al. 1998; Henley et al. 1998). For example, it has been inferred that the bradykinin 2 receptor (BK2R) is sequestered by caveolae (de Weerd & Leeb-Lundberg, 1997). It is not known if caveolin might serve as an adaptor in this process. In addition, some GPCRs, such as the M₁, M₃ and M₄ muscarinic receptors (M1, M3, M4 mAChR), may be endocytosed via a dynamin-dependent process that uses as yet unknown adaptors and/or unknown endocytic machinery (Lee et al. 1998). C, internalization of GPCRs via dynamin-independent pathways has been demonstrated for angiotensin II-1A (AngIIR), M₂-muscarinic (M2 mAChR) and D₂ dopamine (D2R) receptors (Zhang et al. 1996a; Pals-Rylaarsdam et al. 1997; Vickery & von Zastrow, 1999). The cellular machinery for this process is not yet known and needs to be identified in the future.