PArrestin bindingmediated GPCR endocytosis

p-Arrestins act as endocytotic adaptor proteins targeting GPCRs to clathrin-coated pits (Figure 123, Figure 124). To this end, they bind with high affinity to clathrin as well as to the p2-adaptin subunit of the heterotetrameric AP-2 adaptor complex. When present in the clathrin-coated pits, the GPCRs may be internalized (also called sequestered) within the cell by an endocytotic process. This process is dependent on dynamin, a large GTPase that is involved in the pinching off of clathrin-coated vesicles from the plasma membrane. In this respect, it seems that GPCR endocytosis is mediated by the same molecular intermediates as those involved in the endocytosis of receptor tyrosine kinases, except that GPCRs seems to use a common proximal intermediate, P-arrestin.

Advances in receptor visualization techniques have greatly contributed to our understanding of the cellular trafficking of GPCRs and other receptors. Several approaches are possible, such as:

• Transmission electron microscopy with an agonist-colloidal gold complex (Figure 124).

• Transfecting cells with the DNA coding for epitope-tagged receptors (e.g. flag epitope DYKDDDD at the extracellular N-terminus) in combination with immunofluorescence microscopy using anti-flag antibodies and fluorescently labelled secondary antibodies.

• Transfecting cells with the DNA coding for an amino- or carboxyl-terminal recep-tor/GFP fusion protein in combination with fluorescence microscopy (Figure 125).

• The internalization of GPCRs via clathrin-coated pits appears to be effectively inhibited by hypertonic concentrations of sucrose, by low temperatures, by concanavalin A and by the depletion of intracellular ATP and K+.

Ligand (agonist)

Figure 124 Left: Graphic representation of cLathrin coated pits and vesicle. Reprinted from Geoffrey M.Cooper, The Cell: A Molecular Approach 4th edn., © (2007), with permission from ASM Press, Washington DC. Right: Presence of angiotensin II-colloidaL gold complexes on coated pits and (insert) within large endosomes (arrows) at variable depths within the cytoplasm of rat aortic vascular smooth muscle cells (Anderson et al., 1993, reproduced by permission of the American Physiological Society).

Ligand (agonist)

Figure 124 Left: Graphic representation of cLathrin coated pits and vesicle. Reprinted from Geoffrey M.Cooper, The Cell: A Molecular Approach 4th edn., © (2007), with permission from ASM Press, Washington DC. Right: Presence of angiotensin II-colloidaL gold complexes on coated pits and (insert) within large endosomes (arrows) at variable depths within the cytoplasm of rat aortic vascular smooth muscle cells (Anderson et al., 1993, reproduced by permission of the American Physiological Society).

Time (min)

Figure 125 Confocal imaging (fluorescence microscopy) and time-dependence (right) of internalization of enhanced green fluoresccent protein (EGFP)-coupled AT receptor in CHO cells: receptor fluorescence is green when inside the cell and provides a yellow pseudocolor when it colocalizes with the red fluorescence of wheatgerm agglutinin-Texas Red at the cell surface. In non-stimulated cells, the majority of the receptors are at the surface but 30-40% is already cytoplasmic. After 10 min angiotensin II stimulation, 70-75% of the receptors are cytoplasmic. Reproduced from Holloway et al., 2002, Molecular Pharmacology, 61, 768-777.

Time (min)

Figure 125 Confocal imaging (fluorescence microscopy) and time-dependence (right) of internalization of enhanced green fluoresccent protein (EGFP)-coupled AT receptor in CHO cells: receptor fluorescence is green when inside the cell and provides a yellow pseudocolor when it colocalizes with the red fluorescence of wheatgerm agglutinin-Texas Red at the cell surface. In non-stimulated cells, the majority of the receptors are at the surface but 30-40% is already cytoplasmic. After 10 min angiotensin II stimulation, 70-75% of the receptors are cytoplasmic. Reproduced from Holloway et al., 2002, Molecular Pharmacology, 61, 768-777.

• The rapid dissociation of certain agonist- receptor complexes (e.g. AT1 receptors) in an acidic environment has been exploited to differentiate agonists which are bound to cell surface receptors, from internalized agonist molecules. Whereas the former readily dissociate upon mild acid treatment of the cells, the latter will remain in the cell (Figure 126).

Incubation time iminutes)

Figure 126 Incubation of AT1-receptor-transfected CHO cells at 37 °C with [3H]angiotensin II: evolution of acid-sensitive and -resistant binding with time. Reprinted from British Journal of Pharmacology, 126, Vanderheyden, P.M.L., Fierens, F.L.P., De Backer, J.-P., Frayman, N. and Vauquelin, G., Distinction between surmountable and insurmountable selective ATI receptor antagonists by use of CHO-K1 cells expressing human angiotensin II ATI receptors, 1057-1065, © (1999).

Incubation time iminutes)

Figure 126 Incubation of AT1-receptor-transfected CHO cells at 37 °C with [3H]angiotensin II: evolution of acid-sensitive and -resistant binding with time. Reprinted from British Journal of Pharmacology, 126, Vanderheyden, P.M.L., Fierens, F.L.P., De Backer, J.-P., Frayman, N. and Vauquelin, G., Distinction between surmountable and insurmountable selective ATI receptor antagonists by use of CHO-K1 cells expressing human angiotensin II ATI receptors, 1057-1065, © (1999).

Gpcr Endocytosis

Figure 127 Molecular mechanism of (homologous) GPCR regulation: recycling or downregula-tion of internalized receptors. 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. 1, 455-465. Copyright (2002).

Figure 127 Molecular mechanism of (homologous) GPCR regulation: recycling or downregula-tion of internalized receptors. 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. 1, 455-465. Copyright (2002).

Internalization of GPCRs occurs more slowly than desensitization (within seconds), happening over a period of several minutes after agonist exposure. This process is important for receptor resensitization (i.e. recycling to the membrane) and down-regulation (receptor degradation) (Figure 127). The 'sorting' between these two processes will depend on the stability of the GPCR-P-arrestin complex:

Figure 128 Confocal imaging (fluorescence microscopy) of HA-epitope-tagged a1B-adrenergic receptors (revealed with a rhodamine-coupled antibody against the HA-epitope) and green fluo-resccent protein (GFP)-coupled p-arrestin 1 in HEK-293 cells after 15 min agonist exposure (Tohgo et al., 2003, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

Figure 128 Confocal imaging (fluorescence microscopy) of HA-epitope-tagged a1B-adrenergic receptors (revealed with a rhodamine-coupled antibody against the HA-epitope) and green fluo-resccent protein (GFP)-coupled p-arrestin 1 in HEK-293 cells after 15 min agonist exposure (Tohgo et al., 2003, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

HA-receptor GFP-|iarr2 overlay

Agonist-treated

AT1AR

Figure 129 Confocal imaging (fluorescence microscopy) of AT1 receptors and P-arrestin 2 in HEK-293 cells after 15 min agonist exposure (Tohgo et al., 2003, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

• Class A GPCRs (e.g. P2- and a1B-adrenergic receptors, ^-opioid receptors...) recruite P-arrestin 2 more efficiently than P-arrestin 1. They rapidly dissociate from P-arrestin upon internalization. Whereas P-arrestin recycles to the cytosol, the receptors are trafficked to an acidified endosomal compartment (Figure 128) wherein the ligand is dissociated and the receptor dephosphorylated. The receptors are subsequently recycled to the plasma membrane. Receptor recycling is a constitutive process. This implies that, whereas the rate of endocytosis is dependent on the agonist concentration, the rate of recycling is not. Typical recycling takes place with a half-life in the range of 6 to 12 min. However, GPCR recycling to the cell surface can be slowed down by substances, like monensin and nigericin, which raise the pH within endosomes.

• Class B GPCRs (e.g. AT1 and neurokinin NK-1 receptors) recruite both P-arrestins equally well and form stable complexes (Figure 129). These receptors accumulate in endocytic vesicles and are either slowly recycled to the membrane via as yet poorly defined routes or targeted for degradation in lysosomes. This latter process occurs over a period of hours to days. The thrombin receptor and proteinase-activated receptor-2 represent an interesting case since their extracellular N-termini are cleaved during their activation by thrombin and trypsin, respectively. This activation is permanent, which means that the cleaved receptors cannot be recycled to the plasma membrane in their native state. Hence, they need to be sorted to lysosomes for degradation.

Most studies do suggest that, under physiological conditions, the predominant pathway for GPCR endocytosis is P-arrestin- and dynamin-dependent. However, the precise mechanism(s) by which all GPCRs internalize remains a controversial topic and some findings suggest that alternative routes of GPCR internalization could take place. Some internalization could be caveolin-dependent (see Section 4.10).

The physiological importance of receptor resensitization in the maintenance of normal tissue homeostasis is obvious since prolonged or irreversible receptor desensitization would leave a cell unable to respond appropriately to extracellular stimuli. Just as GPCR desensitization provides a mechanism for protecting cells against receptor overstimulation, GPCR resensitization protects cells against prolonged receptor

HA-receptor GFP-|iarr2 overlay

Figure 129 Confocal imaging (fluorescence microscopy) of AT1 receptors and P-arrestin 2 in HEK-293 cells after 15 min agonist exposure (Tohgo et al., 2003, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

desensitization. It has therefore been suggested that receptors that do not recycle will mediate transient responses to agonists, whereas those that are efficiently recycled mediate persistent responses. In this respect, the degree of receptor internalization depends on the ratio between the rate constant for its endocytosis (ke) and the rate constant for its exocytosis/recycling (kr): i.e.% internalized = 100 X ke/ (ke + kr):

The term down regulation refers to a persistent loss of receptors after long-term exposure to agonists. From a physiological viewpoint, cells are rarely exposed continuously to hormones or neurotransmitters, since efficient mechanisms exist to remove them from the extracellular fluid. However, down regulation may occur under pathological circumstances, e.g. when there is continuous secretion of hormones and neurotransmitters from tumours. Down regulation is also important during long-term administration of receptor agonists for therapeutic reasons, since it may be responsible for tolerance. GPCR down regulation occurs as a result of:

• Targeting of internalized receptors to lysosomes. In this respect, continuous agonist exposure may cause multiple rounds of endocytosis and recycling. Even if only a minor portion if the internalized receptors is targeted to the lysosomes during each cycle, this process will finally deplete the cell of a substantial amount of its receptors.

• The second component of receptor down regulation is decreased receptor synthesis. This may be a result of reduced gene transcription or of a post-tran-scriptional event, such as mRNA destabilization. Receptor mRNA destabilization is the prevailing mechanism after long-term agonist stimulation of, for example, p2-adrenergic receptors.

Finally, it has been demonstrated that three different CAMs of the AT1 receptor are constitutively internalized and recycled. This constitutive internalization should be clearly distinguished from the accelerated degradation (down regulation) of misfolded CAMs.

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