Receptor phosphorylation

Free P-y subunits are only found in the plasma membrane at sites of receptor activation. They are able to recognize the C-terminus of 'G protein-receptor kinases' GRK2 and GRK3. This allows the rapid translocation of these kinases from the cytosol to the plasma membrane and provides an extremely precise mechanism for targeting GRK2 and GRK3 to activated GPCRs.

GRK2 and GRK3 were initially termed 'P-adrenergic-receptor kinases' PARK1 and pARK2. Other members of the GRK family have now also been discovered (Figure 119). The family comprises seven family members that share significant sequence homology. Each of the GRKs shares a similar functional organization with a central catalytic domain, an amino-terminal domain that is thought to be important for substrate recognition, and a carboxyl-terminal domain that contributes to the plasma membrane targeting of the kinase. Only GRK2 and GRK3 are attracted by free P -y subunits. Other members may be palmitoylated on carboxyl-terminal cysteine residues (i.e. GRK4 and GRK6) and exhibit substantial membrane localization even in the absence of GPCR activation by agonist. Many factors have been found to regulate GRK activity (Table 14, Figure 120)

GRKs preferentially phosphorylate receptors that are in the agonist-occupied conformation. This phosphorylation occurs at both serine and threonine residues localized within either the endo3 loop or carboxyl-terminal tail domains (Figure 121). No distinct GRK phosphorylation consensus motifs have been identified, but localization of acidic amino acid residues proximal to the site of phosphorylation seems to

Figure 119 Functional domains of the different GRK family members. Reproduced from Ferguson, S. S. G. (2001), Pharmacological Reviews, 53, 1-24, with permission from the American Society for Pharmacology and Experimental Theraputics.

favour GRK2-mediated phosphorylation. Furthermore, GRK phosphorylation alone has little effect on receptor-G protein coupling. However, it stabilizes a conformational state required to promote the interaction of GPCRs with P-arrestins (P-arrestin 1 or P-arrestin 2) (Figure 122). In other words, GRK phosphorylation will increase the

Table 14 GRK activity seems to be regulated by inositol phosphate binding as well as by a complex series of protein phosphorylation events. Reproduced from Ferguson, S. S. G. (2001), Pharmacological Reviews, 53, 1-24, with permission from the American Society for Pharmacology and Experimental Theraputics.

Characteristics of GRK family members

Covalent

Characteristics of GRK family members

Covalent

Family Name

Size (kDa)

Modification

Activators

Inactivators

GRK1 (rhodopsin

63

Farnesylation

Polycations

Recoverin

kinase)

GRK2 (PARKl)

79

N.D.

GPY, PIP2,

MAPK

PKC, c-Src

GRK3 (PARK2)

80

N.D.

GPY, PIP2

N.D

GRK4

66

Palmitoylation

N.D.

N.D

GRK5

68

N.D

Polycations,

PKC,

PIP2

calmodulin

GRK6

66

Palmitoylation

Polycations

N.D.

GRK7

62

N.D. (Farnesylation?)

N.D.

N.D.

Figure 120 Complex regulation of GRK2/3 activity. Reprinted from Trends in Pharmacological Science, 24, Willets, J. M., Challiss, R. A. and Nahorski, S. R., Non-visual GRKs: are we seeing the whole picture?, 626-633. Copyright (2003), with permission from Elsevier.

Figure 121 The central terminus of the AT1 receptor (with residues T332-S338) is the major site for Ang Il-mediated phosphorylation and is the binding site for p-arrestin. Reprinted from Trends in Endocrinology and Metabolism, 14, Thomas, W. G. and Qian, H., Arresting angiotensin type 1 receptors, 130-136. Copyright (2003), with permission from Elsevier.

Figure 121 The central terminus of the AT1 receptor (with residues T332-S338) is the major site for Ang Il-mediated phosphorylation and is the binding site for p-arrestin. Reprinted from Trends in Endocrinology and Metabolism, 14, Thomas, W. G. and Qian, H., Arresting angiotensin type 1 receptors, 130-136. Copyright (2003), with permission from Elsevier.

Figure 122 Putative domain architecture of the P-arrestins. Reproduced by permission of the Company of Biologists, Journal of Cell Science, 115, LuttreLL, L. M. and Lefkowitz, R. J., The role of beta-arrestins in the termination and transduction of G protein-coupled receptor signals, Fig. 2, 455-465, Copyright (2002).

Figure 122 Putative domain architecture of the P-arrestins. Reproduced by permission of the Company of Biologists, Journal of Cell Science, 115, LuttreLL, L. M. and Lefkowitz, R. J., The role of beta-arrestins in the termination and transduction of G protein-coupled receptor signals, Fig. 2, 455-465, Copyright (2002).

affinity of the receptor for P-arrestins (e.g. 10-fold increase in affinity for the GRK2-phosphorylated p2-adrenergic receptor). P-Arrestin binding will only be substantial after GRK phosphorylation for most of the GPCRs. However, depending on the P-arrestin concentration and its binding affinity for the GPCR of interest, substantial p-arrestin binding to the agonist-activated receptor may occur even in the absence of GRK-mediated phosphorylation. Alternatively, certain receptors (e.g. the AT2 angiotensin receptor and P3-adrenergic receptor) do not serve as substrates for GRK and do not bind P-arrestins.

P-Arrestin binding sterically precludes coupling between the receptor and heterot-rimeric G proteins. This leads to the termination of the G protein-mediated signalling (Figure 123). The receptor becomes 'desensitized' (i.e. it can no longer relay an external

A Hgonisl Desensitization frrTTl aHeclor -A , A elathrln

. ) i ) v^i^l'™"« ivi i ^ tt phosphate • ___ ___' L _- jUD i L

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