This page attempts to cover the function of GRKs and arrestins regarding receptor desensitisation. The diverse multi-functionality of arrestins have been well-documented and reviewed variously, notably by the group of Prof. RJ Lefkowitz who has been studying this area for a few decades. Thankfully, many of their studies are publicly available to read.
This summary aims to review intermolecular interactions involving GRKs and arrestins in regulating receptor activity, as well as to present a few relevant numerical values from past studies. The information could be employed in computational studies, in which each particular cellular system involving these are to be dissected, or in which behaviours of each receptor system are to be predicated. For such purposes, any experimentally determined numbers need to be taken carefully to apply unless its uniformity is confirmed in multiple systems. As in any typical biological systems, there might well be unknown/unestablished variables affecting the values. These numbers, however, are highly useful in constructing a crude model which is to be tested vigorously from various dimensions and to be developed into a better representation by incorporating findings from additional studies as necessary.
Note: arrestin-1≡visual arrestin, arrestin-2≡β-arrestin1, and arrestin-3≡β-arrestin2.
Arrestins are named here in the former simple numbered form simply for an ease of typing; however, the latter naming is a more widely applied form in this field of study.
Abbreviations: 5HT (5-hydroxytryptoamine), AlF4- (aluminium fluoride), [Ca2+]i (intracellular calcium concentration), ERK (extracellular signal regulated kinase), ICL (intracellular loop domain), InsP (inositol phosphate), mAChR (muscarinic acetylcholine receptor), NCS (neuronal calcium sensor), PKA (cAMP-dependent protein kinase), PKC (protein kinase C), P-Rho* (active rhodopsin with at least one phosphorylation at its intracellular domain), Rho* (active rhodopsin), TM (transmembrane domain), VILIP (vicinin-like protein)
GRKs (G protein-coupled receptor kinases)
Three subfamilies of GRKs (alternative names)
GRK1 subfamily: GRK1 (rhodopsin kinase), GRK7
GRK2 subfamily: GRK2 (β-ARK1), GRK3 (β-ARK2)
GRK4 subfamily: GRK4 (IT-11 kinase), GRK5, GRK6
Figure 1. Tetrameric complex of GRK2, Gαq and Gβ/Gγ dimer (PDB: 2BCJ, Tesmer VM et al. 2005):
Bovine chains, except the chimeric Gα comprising N-terminal helix of rat Gi1 (1-28) and murine Gq (31-353). Figure below shows GDP-bound chimeric Gi1-Gq (red) associating with RGS homology (RH) domain of GRK2 (blue); shown in turquoise is Gβ and in lime green is Gγ.
GRKs might associate with a 7TM barrel with its short helical region (as seen at the top of this figure) whilst phosphorylating the intracellular regions of the receptor, possibly inducing wider opening of the helical bundle towards intracellular, receptor then releases GRK as the receptor favours arrestin binding
Function of GRKs
GRK2 mediates rapid desensitisation of β2-adrenoceptor
Kinetics analysis performed on permealised A431 epidermoid carcinoma cells revealed that isoproterenol induces phosphorylation of β2-adrenoceptor with a half-time of < 20 s; similarly, β2-adrenoceptor desensitised with a half-time of < 15 s with reduced adenylyl cyclase activity by about 60 % (Roth et al. 1991).
The receptor specific effect of GRK3 and arrestin-3
Coexpression of GRK3 and arrestin 3 in Xenopus oocyte synergistically evoked a rapid desensitisation of DOR and β2-adrenoceptor with a half-time < 4 min, but not for MOR which required 2-3 h of agonist treatment for a similar synergistic effect to be seen (Kovoor et al. 1997).
GRK4 is constitutively active
GRK4 subfamily members have constitutive activity at various degrees, with GRK4 the highest; it can induce receptor internalisation without agonist stimulation (Menard et al. 1996; Rankin et al. 2006).
GRK4 has at least 4 splice variants
GRK4α (full lengths)
GRK4β (without exon 2 with PIP2 binding domain & the beginning of RH domain)
GRK4γ (without exon 15 with the end of RH domain)
GRK4δ (without exons 2 & 15) - lacking RH domain, the least effective negative regulator at least for cAMP signalling (Premont et al. 1996).
GRK5 phosphorylation reduces receptor association with PSD-95 at synapsis
Coexpression study in COS-7 cells showed that isoproterenol-induced GRK5 phosphorylation decreases β1-adrenoceptor associating with postsynaptic density protein PSD-95, which associates the receptor with other synaptic proteins such as ionotropic glutamate receptor, NMDA-R (N-metyl-D-aspartate) (Hu et al. 2002).
GRK6A has palmitoylation site and nuclear localisation signal
At the C-terminal region GRK6A contains multiple elements which could either promote or prevent its localisation at the phospholipid bilayers. It has palmitoylation sites which favours its association with membranes, and it also has a potential nuclear localisation signal. In the region it also has negative acidic residues favouring against associating with membrane, thereby acting as an electrostatic switch which appears to determine its localisation (Jiang et al. 2007). The palmitoylated GRK6 was shown to increase activity ~ 10-fold at phosphorylating β2-adrenoceptor (Stoffel et al. 1998)
Non-redundant roles of GRK subtypes: GRK3 for receptor internalisation, GRK6 for MAPK signal
CCR7 - In response to agonists, chemokines CCL19 and CCL21, CCR7 could recruit different GRKs for each; although an introduction of GRK2 siRNA had no prominent effect for neither agonists, GRK6 siRNA reduced arrestin recruitment by both agonists; GRK3 siRNA significantly reduced CCL-19 mediated phosphorylation (Zidar et al. 2009). As CCL19 was shown to cause receptor desensitisation (Yoshida et al. 1998) and internalisation (Bardi et al. 2001; Byers et al. 2008), its capability of recruiting GRK3 might be the key. The study by Zidar et al. also demonstrated that CCR7 recruiting GRK6, but not GRK3, induces MAPK signalling.
GRK subtypes on family B GPCR in HEK293 cells
SCTR - when secretin receptor was transiently expressed in 293 cells, [125I]secretin bound to the receptor with Kd of 2 nM, generated cAMP signal with EC50 of 0.4 nM, reached maximal response by 20 min and desensitised rapidly thereafter. Agonist-dependent receptor phosphorylation occurred with an EC50 of 14 nM. Pretreating with either PKA- or PKC-inhibitor did not alter cAMP signal. GRK2 and 5 caused a 40% reduction in the maximal cAMP response, with GRK5 causing a right shift in the EC50. GRK4 and 6 did not alter the signal, and GRK3 showed a moderate effect (Shetzline et al. 1998).
Receptor phosphorylation sites by GRKs: C-terminal tail and ICL3
GRK1, GRK2 and GRK5 were shown to target the C-terminal tails of rhodopsin and β2-adrenoceptor (Premont et al. 2004; Palczewski et al. 1991), whereas M2 mAChR was phosphorylated at the ICL3 by GRK2 (Nakata et al. 1994). Phosphorylating one or two sites are sufficient to induce physiological effects (Bennett & Sitaramayya 1988; Ohguro et al. 1993).
The effects of GRKs on regulating receptor activity
A2R - The reduction of GRK2 expression by antisense rat GRK2 stably expressed in NG108-15 cells showed increased cAMP accumulation in response to agonist-induced A2 adenosine receptor activation without affecting A2R expression, but neither by agonist activation of IP-prostanoid nor by secretin receptors (Willets et al. 1999).
α2A-adrenoceptor - After 30 min exposure to epinephrine in CHW cells, the ability of the receptor to activate Gi/0 was decreased by about 78%; the short-term desensitisation was absent when GRK2 inhibiting heparin was introduced, whereas PKA inhibitor showed no effect. The mutant α2A-adrenoceptor lacking S/T phosphorylation sites at its ICL3 showed reduced phosphorylation by 90% in response to agonist-stimulation, failed to undergo short-term desensitisation whilst retaining its expression level (Liggett et al. 1992).
AT1aR - in HEK293 cells, Gq-coupled angiotensin II 1a receptor (AT1aR) exhibited a transient diacylglycerol response in a range of 2-5 min, following agonist-stimulation to complete desensitisation. The event observed depended on GRK2 activity but not on GRK3, -5, or -6, nor arrestin expression (Violin et al. 2006).
CB1R - Two residues, S462 and S430 at the C-terminal region, can be phosphorylated by GRK3 and recruit arrestin-3; these are shown to be involved in agonist WIN55,212-2 induced desensitisation of CB1 in Xenopus oocyte system. GRK3 phosphorylation at the two Ser residues was shown not to be necessary for CB1 internalisation in AtT20 corticotrophs stably expressing CB1 (Jin et al. 1999).
CXCR1R - interleukin-8 induces CXCR1 internalisation in RBL-2H3 rat basophilic leukaemia cells; however in HEK293 cells, which lacks GRK2 (table 2), the process requires coexpression of GRK2 and arrestins. The internalisation in RBL-2H3 was prevented by coexpression of dominant negative V53D-arrestin-2, as well as by dominant negative K44A-dynamin (Barlic et al. 1999).
ETR - Desensitisation of endothelin A and B receptors (ETAR, ETBR respectively) occurred within 4 min in A10 cells expressing ETAR or HEK293 cells expressing either subtype. In HEK293 cells corresponding receptor phosphorylation was observed; it was not affected by PKC inhibitor but reduced 40% by a dominant negative GRK2 mutant. In HEK293, overexpression of GRK2,-5, or -6 enhanced agonist-induced ETR phosphorylation ~ 2-fold. GRK5 inhibited ETR signalling by ~ 25%, whilst the effect by GRK2 was more prominently ~ 80% (Freedman et al. 1997).
M3 mAChR - The reduction of either GRK2, GRK3, GRK6 (but not GRK5), arrestin-2 or -3 increased carbachol-mediated calcium mobilisation in HEK293 cells (Luo et al. 2008).
SMO - The activity of Smoothened (SMO) is tonically inhibited by Patched (PTC); the latter frees SMO upon Sonic hedgehog (SHH) binding, which induces GRK2 phosphorylation of SMO and arrestin-3 recruitment, promoting SMO transport or endocytosis, thereby mediating its signalling (Chen et al. 2004; Meloni et al. 2006).
GRKs have RGS domain
GRKs contain a domain homologous to RGS domain at the N-terminal domain. Likewise to RGS domains which binds to Gα in a manner dependent of AlF4-, Gq/11 from bovine extract was shown to bind to GRK2 and GRK3 in the presence of AlF4-, whereas Gs, Gi, G12/13 of the extract did not. The study demonstrated weak GAP (GTPase activating protein) activity of GRK2 to Gq in the presence of coupling receptor. On contrary GRK2 inhibited Gq-mediated PLCβ activation both in vitro and in cells (Carman et al. 1999b). Though the signalling of other Gq/11 family member G14 was also inhibited by GRK2, it did not have effects on G16 (Day et al. 2003).
GRK2 and Gβγ subunits
Gβγ subunits activate GRK2. By associating via its C-terminal region, GRK2 can also regulate Gβγ signalling by sequestering them. GRK2 can also bind to Gβγ via its N-terminal region that does not overlap with the RGS domain. Gβγ binding to the N-terminal region of GRK2 was shown to enhance its kinase activity to rhodopsin (Eichmann et al. 2003).
Phosphorylation on GRKs
GRK1, GRK5 and GRK6 share conserved Ser/Thr pair at the C-terminal region, which are phosphorylated in the former two; GRK4 has Ser/Ala whereas the motif is absent in GRK2 subfamily. Ala-substitution of the Ser (S488) in GRK1 caused a 20-fold reduction in Km for ATP as well as displaying altered association with rhodopsin (Ohguro et al. 1996).
GRK1 phosphorylation of rhodopsin and autophosphorylation
Km for GRK1 phosphorylation of photo-activated rhodopsin (Rho*) was measured to be 4 (±2) μM, with varying [Rho*] from 0.2 to 20 μM at [ATP] 100 μM; phosphorylation stoichiometry was 1 mol of inorganic phosphate (Pi) per mol of Rho*. Km for GRK1 autophosphorylation was 7 (±2) μM and Vmax was 450 (±52) μM, with varying [ATP] from 0.2 μM to a value ~ 20-fold estimated Km at a [Rho*] 20 μM (Palczewski et al. 1995).
GRK1 autophosphorylates (3-4 sites), preferentially utilising ATP over GTP. The optimum pH for autophosphorylation was higher (pH 7.5 - 8.5) than that of rhodopsin phosphorylation which was around pH 7 (Palczewski et al. 1995). Ki for ATP in the autophosphorylation was 2.6 μM and for GTP was 1.1 mM (Buczylko et al. 1991).
The regulation of GRK2 and GRK5 activity by PKC
GRK2 and GRK5 can be phosphorylated by PKC. Although PKC phosphorylation favours GRK2 to be active by removing inhibition by calmodulin (CaM), the activity of GRK5 can be inhibited by PKC phosphorylation (Krasel et al. 2001).
Negative regulation of GRK2 activity by ERK
GRK2 can be phosphorylated at its C-terminal S670 by ERK for negative regulation affecting its ability to phosphorylate receptors; the phosphorylated GRK2 is less likely be activated by Gβγ (Pitcher et al. 1999).
Negative regulation of GRKs
Recoverin family proteins
The family consists of 14 members in mammals, including: neuronal calcium sensor 1 (NCS1), hippocalcin, neurocalcinδ, visinin-like protein (VILIP) 1-3, recoverin, guanylyl cyclase-activating protein (GCAP) 1-3, and potassium channel-interacting protein (KChIP) 1-4. Some of these are found in the cytosol but some including recoverin, hippocalcin, and NCS1 tend to associate with lipid bilayers as these are myristoylated. In hippocalcin, Ca2+ binding induces conformational change to expose buried myristoyl tail, enabling its association with lipid bilayers at higher Ca2+ concentration (O’Callaghan et al. 2003). On the other hand, NCS1 is more likely be found at the bilayers because its myristoyl tail is constantly exposed (Ames et al. 2000). As for Ca2+ competitions, the affinity of the ion for NCS1 is ~ 10-fold higher compared to CaM (Cox et al. 1994).
In retina
A 24kDa acylated protein, recoverin, was shown to negatively regulate GRK1 (Dizhoor et al. 1993). Ca2+-bound recoverin binds to the N-terminal region of GRK1. Although the binding site of recoverin on activated rhodopsin (Rho*) partially overlaps with that of GRK1, the inhibitory effect of recoverin is not due to the competition for the site; the recoverin-GRK1 interaction rather, disrupts functional conformational change of Rho* inducible by GRK1 upon its binding to the C-terminal region of Rho* (Komolov et al. 2009). Recoverin appears to function more efficiently at the membrane, as IC50 of myristoylated recoverin to GRK1 was 0.8 μM, whilst the comparative value for nonacylated recoverin was ~ 10-fold larger at saturating concentration of Ca2+. Several other members of this family, including NCS1, VILIP1, and hippocalcin, could also bind and inhibit GRK1 (Iacovelli et al. 1999). In intact rod cells, accelerated Rho* inactivation is induced by background light; however, the effect was absent without recoverin. This indicates the role of recoverin in modulating activation period of Rho* in Ca2+-dependent manner (Chen et al. 2010).
In neuronal cells
D2R - In HEK293 cells NCS1 was found to attenuate dopamine-induced internalisation of D2R and D3R initiated by GRK, with more pronounced effect on D2R; the effect coincided with an increase in D2R-mediated cAMP inhibition via Gi. NCS1 was found to colocalise with D2R at sites of synaptic transmission near intracellular calcium stores in striatal neurones. NCS1 was shown to coimmunoprecipitate with either GRK2 or D2R extracted from striatal neurones. These findings indicate that desensitisation of D2R can be mediated by NCS1 by interfering D2 phosphorylation by GRK2. As HEK293 endogenously express NCS1, the study quantified NCS1 level after transient transfection: a 10-fold increase in NCS1 was reported (Kabbani et al. 2002). Modest over-expression of NCS1 in dentate gyrus of adult mice increased spatial memory and promoted exploratory behaviour. Long-term potentiation in the medial perforant path was facilitated; a rapid acquisition of spatial memory was also observed. The effects were reversed by application of either a cell-permeant peptide designed to disrupt NCS1 binding to D2R, or a D2R-selective antagonist L-741,626 (Saab et al. 2009).
β2-adrenoceptor - In C6 glioma cells VILIP did not significantly influence desensitisation of β2-adrenoceptor. Although an increase in cAMP level was seen, the effect of VILIP likely was on adenylyl cyclase, as VILIP also increased cAMP via forskolin; in contrast, non-myrisoylated VILIP reduced cAMP levels (Braunewell et al. 1997).
Aside: ARF and NCS1 in Golgi apparatus
A GTPase, ADP-ribosylation factor (ARF) proteins have been shown to interact with NCS1 (Haynes et al. 2005). For rapid GTP hydrolysis, ARF requires GTPase-activating protein GAP1 or GRK-interacting protein GIT1 or 2; the latter two were reported to facilitate ARF6 activity (Vitale et al. 2000). Both ARF and NCS1 regulate phosphatidylinositol 4 kinase III β (PI4Kβ), which recruit trafficking components to the trans-Golgi network (TGN) and regulate traffics from the TGN to the plasma membrane; NCS1 independently produce bidirectional effects to activate PI4Kβ, or inhibit activation by ARF1 by recruiting GDP-bound ARF1 at high calcium concentration (Haynes et al. 2005). Association of ARF1 and NCS1 function to negatively regulate exocytosis at Golgi apparatus. ARFs 1,3,5 and 6 have been shown to interact with NCS1, but only ARF1 and 3 can interact with PI4Kβ; the stimulatory effect on PI4Kβ was only seen with ARF1 but not with ARF5 or 6 (Haynes et al. 2007).
The expression of mRNAs encoding recoverin family proteins in adult rat brain
Recoverin: the retina, pineal gland
VILIP1: widely distributed (except the caudate-putamen)
NCS1: primary afferent neurones, dendrites of hippocampal pyramidal, granule cells
Hippocalcin: the forebrain, neocortex, hippocampus and caudate-putamen
VILIP2: the forebrain, neocortex, hippocampus and caudate-putamen
VILIP3: cerebellum (Purkinje and granule cells), ventral nuclei throughout the forebrain and midbrain, the medial habenulae, the superior and inferior colliculi
Calmodulin (CaM)
Recoverin-GRK1 interaction can be competed by CaM, which also negatively modulate GRKs. CaM was shown to bind to the C-terminal region of GRK5 (Levay et al. 1998). GRK4 subfamily members tend to bind to CaM with higher affinity: IC50 of CaM for GRK5 was in a range of 40-50 nM and for GRK4 was 80 nM , whereas the values for GRK2 and GRK3 was approximately 2 μM (Pronin et al. 1997; reviewed by Iacovelli et al. 1999). CaM could trigger autophosphorylation of GRK5 at distinct sites from the standard autophosphoryaltion sites, in order to decrease its association with receptor (Pronin et al. 1997).
Inhibition of GRKs by calcium-sensing proteins (associations confirmed)
GRK1 (Recoverin, NCS1, VLIP1)
GRK2 (CaM, NSC1 ⚚)
GRK3 (CaM)
GRK4 (CaM)
GRK5 (CaM)
GRK6 (CaM)
GRK7 (Recoverin §)
α-Actinin inhibits GRKs along with CaM and PIP2
α-Actinin is a potent inhibitor of GRKs, modulating substrate preferences of GRKs. In the presence of Ca2+-bound CaM and α-Actinin, GRK5 was shown to favour cytosolic substrates over membrane-bound, whilst phosphatidylinositol 4,5-bisphosphate (PIP2) and α-Actinin reversed the preference (Freeman et al. 1998).
Actin
Actin was shown to inhibit kinase activity of GRK5. GRK5 binds to both actin monomers and filaments, with Kd of 0.6 μM and 0.2 μM, respectively (Freeman et al. 1998).
Coveolin
GRKs were shown to associate with the scaffolding domain of caveolin, in a manner inhibitory to GRKs phosphorylating receptor (Carman et al. 1999a).
Other non-GPCR substrates shown to be phosphorylated by GRKs
Cytoskeletal protein, tubulin was shown to be phosphorylated by GRK2 (Haga et al. 1998; Carman et al. 1998; Pitcher et al. 1998) and by GRK5 (Carman et al. 1998). GRK2 can also phosphorylate receptor tyrosine kinases (Freedman et al. 2002), ribosomal protein P2 which HEK293 endogenously express (Freeman et al. 2002), and 14-kDa proteins known as synucleins which are highly expressed in the brain (Pronin et al 2000). GRK5 was shown to phosphorylate p53 (Chen et al. 2010). GRK5 was also shown to phosphorylates Hsp70 interacting protein (Hip) at its S346; the phosphorylation was reported to be a requisite for effective agonist-induced internalisation of CXCR4 (Barker & Benovic 2011). GRK6A was shown to be responsible for constitutive phosphorylation of NHERF in HEK293 cells (Hall et al. 1999).
Arrestins
GRK1 inactivates rhodopsin whilst arrestin accelerate the process
When the inactivation of Gt on light-scattering as well as of phospodiesterase on cGMP hydrolysis were measured, GRK1 alone showed effects with IC50 of 50 nM for 4.4 μM rhodopsin. In the presence of arrestin-1, metarhodopsin II was inactivated by 1 P attached per rhodopsin, whereas without arrestin-1 it required 12-16 P per rhodopsin (Bennett & Sitaramayya 1988).
Affinity and stoichiometry of arrestins to receptors
High-affinity binding of arrestin to GRK1-phosphorylated rhodopsin requires incorporation of 1 mol of phosphate per 1 mol of rhodopsin (Krupnick et al. 1997a). Monomeric rhodopsin in nanodiscs, light-activated and GRK1-phosphorylated, bind to arrestin-1 with low nM affinity with 1:1 receptor to arrestin stoichiometry (Bayburt et al. 2011); the 1:1 stoichiometry also applies to GRK2-phosphorylated β2-adrenoceptor to β-arrestin.
Affinity of receptors for arrestins
β2-adrenoceptor - The binding affinity of purified arrestin to the receptor purified, reconstituted into lipid vesicles, activated by isoproterenol and phosphorylated, was found to be 1.8 nM (Kd). The study also found that arrestin can bind to receptors dephosphorylated with acid phosphatase, with ~30-fold lower affinity (Söhlemann P et al. 1995).
M2 mAChR - The Kd values for arrestin-1 and arrestin-2 binding to M2 mAChR were 7.2 (±1.2) nM and 0.48 (±.06) nM, respectively (Gurevich et al. 1993).
Arrestins association with CaM and microtubules
Arrestins can bind to Calcium-bound CaM with a Kd of ~ 7 μM. The binding site in the centre of arrestin overlaps with its binding sites for receptor and for microtubules. Arrestins were shown to sequester CaM thereby preventing microtubule to bind; higher receptor affinity in nM range for arrestins easily outcompete CaM (Wu et al. 2006a).
Receptor association with arrestin and CaM
V1aR - ICL3 and/or C-terminal segoment of V1aR engineered onto maltose-binding protein bound to arrestin-2 and CaM with affinities in the μM range (Wu et al. 2006b).
The regions of receptors involved in interacting with arrestins
α-adrenoceptors - Arrestins promote internalisation of α2b- and α2c-adrenoceptors but not of α2a-adrenoceptor. The ICL3 of the former two subtypes were shown to associate directly with arrestin-3, but not with that of the latter, whilst arrestin-2 associated only with the ICL3 of α2b-adrenoceptor amongst the three. Mutagenesis study indicated two arrestin-3 binding domains in the ICL3 of α2b: residues 194-214 and residues 34-368, in the regions approaching transmembrane domains (DeGraff et al. 2002).
β2-adrenoceptor - The C-terminal region (355-364) containing four Ser/Thr residues participate in interacting with arrestin-3 (Krasel et al. 2008).
CB1R - Whilst associating with arrestin-2, phosphorylated C-terminal peptide derived from CB1 (5P:454-473) was shown to adopt a helix-loop conformation in a manner similar to the rhodopsin C-tail peptide (7Ps: 330-348) bound to visual arrestin (Singh et al 2011).
D1R - Both arrestin-2 and -3 from striatal homogenates bound to the C-terminus of D1 dopamine receptor, and the purified forms also bound to ICL3 of the receptor to a lesser extent (Macey et al. 2005).
D2R - D2 dopamine receptor bind to both arrestin-2 and -3 from striatal homogenates via ICL3, whilst the purified arrestins bound to both ICL2 and -3 (Macey et al. 2004). The key region of arrestin interaction is in ICL3 close to TM5 (Lan et al. 2009a).
D3R - D3 dopamine receptor associates with arrestin-3 at its ICL2, albeit with weaker affinity than D2R, partly due to the absence of K149 located at ICL2 nearby TM4 in D2R (Lan et al. 2009b).
OX1R - A Thr/Ser clusters located near the C-terminus of orexin 1 receptor (OX1R) determines the receptor association with arrestin-3 (Milasta et al. 2005).
PTHR - The receptor’s C-tail was shown to determine arrestin binding to the receptor (Vilardaga et al. 2002).
M2 mAChR - Multiple phosphoryaltion of sites within 307-311 in ICL3 favours arrestin binding to M2 mAChR (Lee et al. 2000).
M3 mAChR - Two segments in ICL3, G308-L368 and K425-K497, were required for arrestin-2 association, whereas the latter segment only was sufficient for interacting with arrestin-3 (Wu et al. 1997).
Rhodopsin - phosphorylation of S343 in the C-terminal region of rhodopsin is an absolute requirement for arrestin-1 binding (Zhang et al. 1997). ICL3 was also shown to participate in arrestin binding (Krupnick et al. 1994).
LHR - Luteinizing hormone receptor (LHR) which associated tightly with arrestin underwent agonist-induced desensitisation on adenylyl cyclase activity (Mukherjee et al. 1999a). A synthetic peptide corresponding to the entire ICL3 of the LTR prevented desensitisation with an ED50 of 10 μM. Association between the Sepharose-bound ICL3 peptide and arrestin-2 were confirmed (Mukherjee et al. 1999b).
Y2R - Neuropeptide Y2 receptor has a distal C-terminal motif (373-379) which is associated with arrestin-3 (Walther et al. 2010).
The regions of arrestins involved in interacting with receptors
Rhodopsin - A cationic region of bovine arrestin1 (V170-R182) involves in binding to P-Rho* (Kieselbach et al. 1994). Two regions in visual arrestin recognise rhodopsin: residues 49-90 in β-strands 5/6 and adjacent N-terminal loops, and 237-268 in the C-terminal β-strands 15/16 (Vishnivetskiy et al. 2004).
M2 mAChR - homologous regions to the above in arrestin-2 were shown to participate in associating with M2 mAChR (Vishnivetskiy et al. 2004). M2 mAChR can be desensitised in the presence of both GRK2 and arrestin-2 (Schlador & Nathanson 1997).
Arrestins could stabilise active conformation of receptors
5HT2AR - 5HT2AR can be desensitised or internalised independently of arrestins in some cell types. Although co-transfection of arrestin-2 had no such effects observed on 5HT2AR, introduction of constitutive active arrestin-2 mutant (Arr2-R169E) decrease agonist efficacy of Gq/11 signalling but increased the potency; moreover, agonist-independent interaction between Arr2-R169E and 5HT2AR was disrupted by inverse agonist (Gray et al. 2003). These results provide an evidence for arrestin-2 in favouring active receptor conformation and their prolonged interaction stabilise such conformation, leading to desensitisation.
The relevance of receptor conformational state to internalisation
PTHR - Parathyroid hormone receptor constructs without either N289 (on TM3) or K382 (on ICL3) exhibited a 30-60 % reduction in internalisation in HEK293 cells. The two residues have been suggested to regulate internalisation of arrestin-bound receptor to the early endosomes in initiating endocytosis (Vilardaga et al. 2002). The evidences which support the importance of TM3 residues in GPCR conformations and activation are abundant elsewhere.
Alternative splicing variants differ in receptor-arrestin interactions and internalisation patterns
TPR - Thromboxane A2 receptor exist in two isoforms, TPα (343 a.a.) and TPβ (407 a.a.); the two are identical up to 328th residue. In HEK293, only TPβ internalise upon agonist-stimulation in the process involving dynamin, GRK and arrestin. The internalisation can be promoted by arrestin-3 over-expressed, but TP truncated at 328 was resistant to the effect. The residues between 355 and 366 of TPβ appears to be critically involved in agonist-induced internalisation (Parent et al. 1999).
In addition, TPβ, but not TPα, undergoes tonic internalisation dependent on dynamin (and temperature), independent of GRK or arrestins. Tonically internalised TPβ was shown to recycle back to the cell-surface (Parent et al. 2000).
Competitors for arrestin-receptor interactions
Competition between arrestin and G-protein for phosphorylated receptors
Competition between arrestin-1 and Gt for the binding to GRK2-phosphorylated rhodopsin (P-Rho*) occurs by IC50 of 50 nM arrestin-1 with a molar ratio of 1.7:1, arrestin to Gt. Maximal displacement (~ IC80) observed was by 200 nM arrestin-1. No effector competition was seen for unphosphorylated Rho* (Krupnick et al. 1997a).
Competition between inositol phosphates and receptors for arrestins
Certain well-phosphorylated forms of inositol were shown to compete for arrestins binding against rhodopsin. Competition binding assays showed Kd for arrestin binding to InsP4 or InsP6 to be 12 μM and 5 μM, respectively. Gt, GRK1, or cGMP phosphodiesterase were not affected by the inositol phosphates (Palczewski et al. 1991).
Time scales for the arrestin-induced desensitisation of receptors
Time scales for arrestin recruitment and subsequent receptor internalisation
NK1R - Upon substance P stimulation in KNRK rat K-ras transformed kidney cells, arrestin-2 was recruited within a minute to the plasma membrane; then SP-bound NK1 and arrestin-2 or -3 were co-localised in the same endosomes (2 - 10 min) and stayed there up to 1h. NK1 was recycled back to the cell membrane in the duration of 4-6 h, whilst Gq/11 remained at the cell membrane (McConalogue et al. 1999).
A longer time scales in agonist desensitisation of A1 adenosine receptor
A1R - It needs a prolonged agonist exposure for desensitisation, from 15 min to several hours or days (Parsons & Stiles 1987; Ramkumar et al. 1991; Green et al. 1992; Longabaugh et al. 1989).
Short term agonist desensitisation of A1 adenosine receptor by Gi/0 uncoupling through actions of GRK and arrestin
Pre-treatment of DDT1MF-2 hamster smooth muscle cell-line with 1μM (R)-PIA, a full agonist, reduced iodinated APNEA binding rapidly down to about 70% after 30 min, followed by a gradual decrease to about 45 % by 4h; at this point, Bmax was reduced to 251 ± 67 from a control 342 ± 82 fmol/mg protein. A high Bmax in the pre-treated was seen in saturation assay with antagonist [3H]DPCPX binding, of Kd was unaffected. The 4h pre-treatment at 37 ˚C roughly halved proportion of G-protein-coupled, high affinity state receptor; expression levels of G-protein subunits were unaffected. The desensitisation was reversed by alkaline phosphatase, phosphatases 1 or 2A treatment. Incubation with (R)-PIA led to rapid translocation of GRK within 1h of exposure. Purified GRK2 showed enhanced affinity for arrestin over Gi/0 (Nie et al. 1997).
Varied time scales in agonist-induced desensitisation
β2-adrenoceptor - In Xenopus oocyte, coexpression of β2-adrenoceptor with GRK3 and arrestin-3 induced a rapid desensitisation with a half-life < 4 min (Kovoor et al. 1997).
DOR - In the study above, δ opioid receptor (DOR) was also rapidly desensitised in the condition as above.
MOR - In the study above μ opioid receptor (MOR) was desensitised more slowly, requiring agonist treatment for 2-3 h.
Functional comparisons of non-visual arrestins: arrestin-2 and -3
Arrestin-2 and arrestin-3 differ in their conformational dynamics upon receptor binding
Arrestin-2 and -3 undergo different conformational changes upon binding to receptor, when their conformation changes induced by phosphopeptide derived from the C-terminus of V2R. The key difference was in the flexibility of the hinge region (Xiao et al. 2004; Nobles et al. 2007).
Arrestin-3 is likely be more effective than arrestin-2
Although arrestin-2 and -3 are structurally close with about 78 % similarly in protein sequence estimated (Attramadal et al. 1992), slight differences in physiological distribution and function have been reported as follows.
Expression level of arrestin-2 and arrestin-3
Northern blog analysis of rat mRNA from various tissues revealed that arrestin-3 showed more prominent expression than arrestin-2, most notably in the spleen. Both arrestins are expressed extensively in the cortices, hippocampus, brain stem, and a slightly less amount in the pituitary (Attramadal et al. 1992).
α2-adrenoceptors - Agonist-induced internalisation in COS-1 cells varied in magnitude among subtypes and was in an order of α2a< α2b< α2c. Coexpression of arrestin-2 or -3 largely enhanced α2b internalisation via clathrin-pits and endosomes, whereas the process was selectively promoted by arrestin-3 for α2c, and only minor effect was seen for α2a by either arrestins. Coexpression of GRK2 had no effect on the internalisation of any subtypes either with or without arrestins. The internalisation was inhibited by K44A-dynamin for α2b and α2c. Neither arrestin-3 nor K44A-dynamin affected p42/p44 MAPK activity (DeGraft et al. 1999).
It has been said for some receptors that arrestin-3 translocate to active receptor more efficiently than arrestin-2 (mentioned in Laporte et al. 1999)
D1R - Agonist treatment for 5 and 20 min caused translocation of arrestin-3 but not of arrestin-2. Internalisation of D1R in a range of 35-45% was maximal after 2-5 min of agonist treatment (Macey et al. 2005).
D2R - Although in neuroblastoma NS20Y cells both arrestin-2 and -3 appeared to induce D2R internalisation (35-45%) which was maximal after 20 min agonist-treatment, in neonatal neurones 2h treatment induced recruitment of arrestin-2 but not of arrestin-3 (Macey et al. 2004).
TSHR - In rat thyroid cell-line FRTL5, the expression of arrestin-3 was undetected but arrestin-2 was expressed in a manner regulated by TSH. When TSHR was co-expressed with GRK2 and/or arrestin-2 in COS-7 cells, TSH-induced cAMP accumulation was reduced by 35-45 % (Lacovelli et al. 1996).
Arrestins associate with diacylglycerol kinases
The activation of Gq-coupled M1 mAChR employs arrestins which exhibit dual functionality in favouring degradation of diacylglycerol by associating with diacylglycerol kinases, as well as inhibiting its production by participating in receptor desensitisation (Nelson et al. 2007).
Arrestin-2 associates with G-protein subunits Gβ1γ2
The association between Arrestin-2 and Gβ1γ2 were shown in vitro. The overexpression of arrestin-2 facilitated Gβ1γ2-mediated Akt phosphorylation, nuclear translocation of NFκβ and its reporter gene activation (Yang et al. 2009).
Arrestin-1 can self-associate to form multimeric complexes
Arrestin-1 from two species, human and mouse, can exist in multimeric equilibrium between monomer, dimer and tetramer (Kim et al. 2011).
Arrestins recruit receptors to clathrin-coated pits when bound to phosphoinositides
High-affinity sites for phosphoinositides were identified in arrestin-2 and arrestin-3. When mutant arrestin-3 which do not bind to phosphoinositides was expressed in COS-1 cells, it neither facilitated β2-adrenoceptor internalisation nor gathered the receptor to clathrin-coated pits (Gaidarov et al. 1999).
Arrestins associations by the cell-surface membrane for receptor trafficking
Arrestin, AP2 & receptor
Arrestin-3 associates with heterotetrameric adaptor protein AP-2, which binds to heavy chain of clathrin and intracellular domain of the receptor. The association between AP-2 and β2-adrenoceptor upon agonist binding peaked at 2 min and decreased after 5 min (Laporte et al. 1999).
Arrestin & ARF6, ARNO
Arrestins can bind to a small GTPase, ADP-ribosylation factor 6 (ARF6), and also to ARF nucleotide exchange factor (ARNO), both of which are components of the receptor endocytic machinery. Agonist-binding to receptor induces arrestin to associate with GDP-bound ARF6 to facilitate ARNO-promoted GTP binding (Claing et al. 2001).
Arrestin & clathrin
Arrestins bind to clathrin (Goodman et al 1996) at its C-terminal domain: in arrestin-3 involving hydrophobic (L373, I374 and F376) and acidic (E375 and E377) residues. An arrestin-3 Ala-mutant lacking the L, I & F was defective in promoting β2-adrenoceptor internalisation in COS-1 (Krupnick et al. 1997b). It has also been shown that both N- and C-terminal ends of arrestin-2 inhibit basal interaction with clathrin (Kern et al. 2009). The plasma membrane facing end of clathrin heavy chain was shown to be required for arrestin recognition; the interaction involves a conserved E89 and relatively conserved K96 and K98 of clathrin (Goodman et al. 1997).
Arrestin & NSF
Arrestin-2 was shown to bind to an ATPase, N-ethylmaleimide-sensitive fusion protein (NSF), with a preference to the ATP-bound form. Overexpressing NSF in HEK293 significantly enhanced agonist-induced internalisation of β2-adrenoceptor (McDonald et al. 1999). Rapidly recycling β2-adrenoceptor was shown to interact with NSF at the C-terminal tail notably involving last three side chains. Isoproterenol-stimulation induced colocalisation of the two, receptor internalisation, and subsequent treatment with antagonist propranolol brought receptors back on the surface; none of these observed for the C-terminally truncated β2-adrenoceptor (Cong et al. 2001).
Arrestin & NHE
Na(+)H(+) exchanger NHE5 can be phosphorylated by acidtropic CK2 at highly acidic, Ser/Thr-rich, di-Ile motif (697-723) in the C-terminal region, for arrestin-3 recognition; the site was less likely be phosphorylated by CK1 or GRK2 (Lukashova et al. 2011).
Others associating arrestins
Interacting proteins with arrestin-2 and -3 are summarised and tabulated by Xiao et al. 2007 (PubMedCentral open access).
Arrestin can be dephosphorylated for endocytosis
In cytoplasm, arrestin-2 is constitutively phosphorylated for it to be recruited upon receptor activation. After associating with the receptor, arrestin-2 can be dephosphorylated rapidly, enabling it to bind clathrin; arrestin-2 can be re-phosphorylated afterwards (Lin et al. 1997).
Receptor recycling requires arrestins
FPR - An investigation on N-formyl peptide receptor (FPR) trafficking in MEF cells revealed that the receptor could internalise in the absence of arrestin-2 or-3, but FPR recycling requires them. In the absence of arrestins, FPR may be trapped in the perinuclear recycling compartment in the cytoplasm (Vines et al. 2003).
Receptor recycling is regulated by ubiquitination and deubiquitination of arrestins
Although sequestration of ERK to microtubules by arrestins decreases the ERK activation, arrestin-binding to Mdm2 to microtubules increase ubiquitination dramatically (Hanson et al. 2007).
V2R - Some receptors, such as β2-adrenoceptor, recycle rapidly; on the other hand, some receptors, such as V2 vasopressin receptor, tend to be internalised and recycle slowly. The process involves ubiquitination of arrestins by E3 ubiquitin ligase, Mdm2. The arrestin-modification can be transient or stable depending on the balance between ubiquitination and deubiquitination, the latter of which appear to cause arrestin to dissociate from its bound receptors, thereby allowing receptor to recycle back (Shenoy & Lefkowitz 2003).
AT1aR - The receptors co-internalised with arrestins can activate ERK1/2 on endosomes. This process requires ubiquitination of arrestin-3 for angiotensin II type 1a receptor (AT1a). Without two ubiquitination sites on arrestin-3, it and AR1a could not integrate into stable endocytic complexes, and ERK1/2 activation was impaired. The ubiquitination profile on arrestin-3, however, appear to differ among receptors regarding their effects on endosomal trafficking (Shenoy & Lefkowitz 2005).
Aside: Time scale of agonist-induced desensitisation in neurones based on [Ca2+] measurement
NK1R - In guinea pig myenteric neurones, SP stimulation induces rapid desensitisation of NK1R. Desensitisation is minimal for brief exposure: stimulating myenteric neurones with 100 nM SP for 1 min caused an increase in [Ca2+]i; the cells were washed and the neurones were re-exposed to 100 nM SP 5 min after the initial exposure; an increase in [Ca2+]i seen was only slightly less than the previous response. However, when the first exposure period was increased to 5 min and then given the second dose 10 min later, there was a strong desensitisation. Desensitisation was detectable after stimulated with 0.1 nM SP, and was maximal with 100 nM, which was sufficient to desensitise the receptor fully at 10 min. Half-maximal desensitisation was seen with ~ 10 nM SP. Resensitisation was half-complete after ~ 22 min; it was completed after 30 min (McConalogue et al. 1998).
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Appendix Table 1 & 2
Expression profiles of endogenous GPCR effectors in four cell types, based on mRNA expression on microarray. Data from Atwood et al. 2011. ND (not detectable), - (trace detected).
Components
|
HEK293
|
AtT20
|
BV2
|
N18
|
Arrestin-2
|
****
|
-
|
*
|
**
|
Arrestin-3
|
********
|
****
|
*****
|
****
|
Gs
|
********
|
***
|
****
|
*****
|
Gi1
|
***
|
ND
|
ND
|
ND
|
Gi2
|
*******
|
**
|
*****
|
******
|
Gi3
|
****
|
ND
|
***
|
****
|
G0
|
-
|
-
|
*
|
***
|
G02
|
**
|
ND
|
ND
|
ND
|
Gq
|
******
|
*
|
**
|
***
|
G12
|
****
|
*
|
**
|
****
|
G13
|
*****
|
ND
|
***
|
****
|
G14
|
**
|
ND
|
-
|
-
|
G15
|
*
|
*
|
*
|
*
|
Gz
|
*****
|
-
|
-
|
**
|
Gβ1
|
*******
|
***
|
*****
|
******
|
Gβ2
|
********
|
**
|
****
|
*****
|
Gβ3
|
***
|
-
|
ND
|
-
|
Gβ4
|
****
|
ND
|
-
|
**
|
Gβ5
|
****
|
*
|
*
|
***
|
Gγ2
|
***
|
-
|
**
|
**
|
Gγ3
|
***
|
-
|
ND
|
ND
|
Gγ4
|
*****
|
ND
|
-
|
***
|
Gγ5
|
*****
|
*
|
**
|
***
|
Gγ7
|
*
|
*
|
*
|
*
|
Gγ8
|
ND
|
*
|
*
|
**
|
Gγ10
|
******
|
*
|
**
|
**
|
Gγ11
|
**
|
-
|
ND
|
ND
|
Gγ12
|
******
|
ND
|
-
|
-
|
Expression profiles of endogenous kinases in four cell types. Data from Atwood et al. 2011. ND (not detectable), - (trace detected).
Kinases
|
HEK293
|
AtT20
|
BV2
|
N18
|
GRK2
|
ND
|
*
|
****
|
**
|
GRK3
|
****
|
-
|
ND
|
**
|
GRK4
|
***
|
ND
|
ND
|
ND
|
AMPK-α1
|
****
|
ND
|
***
|
****
|
AMPK-α2
|
****
|
***
|
**
|
**
|
PKA-1β
|
****
|
-
|
-
|
***
|
PKA-2β
|
****
|
-
|
-
|
***
|
AMPK-β1
|
******
|
**
|
**
|
***
|
AMPK-β2
|
****
|
ND
|
ND
|
*
|
PKA-1α
|
*******
|
ND
|
**
|
******
|
PKA-2α
|
****
|
ND
|
**
|
***
|
AMPK-γ1
|
*******
|
***
|
**
|
***
|
AMPK-γ2
|
****
|
*
|
*
|
**
|
PKA-cα
|
******
|
****
|
***
|
*****
|
PKA-cβ
|
****
|
ND
|
***
|
***
|
PKCα
|
******
|
**
|
-
|
*
|
PKCβ
|
**
|
*
|
-
|
-
|
PKCδ
|
**
|
*
|
*
|
*
|
PKCε
|
****
|
*
|
***
|
-
|
PKCη
|
****
|
*
|
*
|
***
|
PKCι
|
****
|
ND
|
-
|
**
|
PKCθ
|
**
|
ND
|
ND
|
-
|
PKCζ
|
******
|
ND
|
-
|
*
|
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