Criterios Agregados

Activated G-protein coupled receptors are substrates for several families of kinase proteins, which include second messenger-regulated kinases such as PKA and PKC (Hausdorff et a l, 1990), and G-protein coupled receptor kinases (GRKs). The conformational changes which occur as a result of agonist binding and receptor activation expose a number of serine and threonine residues in the C- terminus and the third intracellular loop of GPCRs, which greatly enhances receptor phosphorylation by GRKs. There are currently 6 members of the GRK family (GRK 1-6). The most commonly studied are GRKl, or rhodopsin kinase (Kuhn, 1978), and GRK2, or P-adrenergic receptor kinase, which is now thought to phosphorylate a large number of GPCRs (Krupnick & Benovic, 1998).

Phophorylated receptors are substrates for a family of cytosolic proteins called arrestins (Krupnick & Benovic, 1998). Whilst GRK catalysed phosphorylation of GPCRs alone may modestly desensitise receptor signalling (Bennett & Sitaramayya, 1988) the binding of arrestins to phosphorylated receptor is the primary mechanism for the rapid uncoupling of receptors from G-proteins (Krupnick & Benovic, 1998). Furthermore, receptor phosphorylation and arrestin binding are critical steps in the initiation of internalisation of muscarinic M2

(Tsuga et al, 1994) and the Pz-adrenergic receptor (Ferguson et a l, 1995). The internalisation of surface expressed GPCRs following prolonged agonist exposure was originally considered to be part of the process of receptor down regulation. However, internalisation has also been demonstrated to be critical for receptor dephosphorylation and resensitization of the P2-adrenergic receptor (Yu et a l,

1993; Zhang et a l, 1997). More recently it has been discovered that receptor internalisation is a prerequisite for the G-protein coupled receptor mediated activation of mitogenic signalling pathways (Daaka et a l, 1998; Luttrell et a l,

1999).

Support for the C-terminus of GPCRs as the initial component for ‘scaffolds’ of other proteins comes from the interactions between the p2 receptor and the Na^/FC-exchanger regulatory factor (NHERF, Hall et a l, 1998b). The NHERF family of peptides contain PDZ domains which recognise the C-terminal motif D- S/T-x-L of both the P2-adrenergic receptor and the purinergic P2Yi receptor (Hall

et al, 1998a). However, the precise biological relevance of the NHERF

The secretagogue, type II receptor family contain a group of 7TM receptors whose expression and pharmacological properties are dependent on interactions with accessory proteins termed receptor activity modifying proteins (RAMPs, Foord & Marshall, 1999). It had been recognised that whilst the operational characterisation of secretagogue receptors had demonstrated the existence of at least five members of the family (calcitonin, amylin, calcitonin-gene-related- peptide (CGRP) -1 and -2, adrenomedullin), only two genes could be cloned (the calcitonin receptor and the calcitonin receptor like receptor CRLR, Flühman et al,

1995). Meanwhile, cRNA from CGRP receptor expressing cells was found to induce novel CGRP responses in Xenopus oocytes (McLatchie et a l, 1998). The protein responsible was cloned and termed receptor activity modifying proteins (RAMPl). Two additional RAMPs were cloned (RAMP 2 and 3) and demonstrated to produce adrenomedullin receptors when co-expressed with CRLR (McLatchie et a l, 1998). Amylin receptors can be created by the actions of RAMPs with a calcitonin receptor gene product (Chen et a l, 1997). The biological functions of RAMPs are integral to the functioning of calcitonin related receptors in that they transport the receptors to the cell surface, determine the receptor pharmacology and influence receptor glycosylation states (McLatchie et a l, 1998).

The expression and function of the family HI GPCR gamma-aminobutyric acid

(GABAb) receptor is dependent on the co-expression of two separate 7TM

receptors G A B A bri and GABAbr2 (Mohler & Fritschy, 1999). Native GABAb

receptors regulate potassium and calcium channels via G-protein activation (Bowery, 1993). When expressed alone, G A B A bR I fails to bind agonists with

high affinity or couple efficiently to signal transduction pathways (Kaupmann et a l, 1997). Fully functional recombinant GABAb receptors, with similar pharmacological properties to native receptors, are generated only upon co­ expression of a second GABAb receptor, the GABAbr2 receptor (Jones et a l,

1998; Kaupmann et a l, 1998; White et a l, 1998).

The concept that family A receptors can form dimers and higher order multimers, and that these complexes are important for receptor function, is beginning to emerge. Pharmacological evidence for 7TM receptor heterodimers has been demonstrated for the k and 5 opioid receptors which, when coexpressed, demonstrate ligand binding and functional properties that are distinct from those of either receptor (Jordan & Devi, 1999). Evidence for an interaction between family A 7TM GPCRs at the molecular level has been demonstrated using 0C2- adrenergic and Ms-muscarinic receptors in which TM 6 and 7 have been exchanged (Maggio et a l, 1993). No radioligand binding could be detected when the chimeric receptors were expressed separately. However, when the constructs were coexpressed in the same cell line, binding of both the muscarinic and adrenergic radioligands could be detected. Based on the relative orientations of the transmembrane regions, it is postulated that dimers occur either as contact dimers or ‘domain swapped’ dimers, involving TM5 and 6 (Gouldson et a l,

2000).

Co-immunoprecipitation studies have provided supportive evidence that differentially epitope tagged receptors may exist as homo- (Cvejic & Devi, 1997) and hetero- (Salim et a l, 2002) dimers. In intact cells, resonance energy transfer can be demonstrated between receptors differentially labelled with energy donor

and acceptor molecules (for a review see Salim et a l, 2002). Resonance energy transfer occurs across distances that are typically less than 100Â thus suggesting molecular proximity between different receptors. Both co-immunoprecipitation and resonance energy transfer techniques have demonstrated that agonists are able to perturb the extent of receptor proximity. For the 6-opioid receptor, increasing concentrations of the agonist DADLE decreased the level of the dimer in immunoprecipitates, and correspondingly increased the level of monomer (Cvejic & Devi, 1997). In contrast, using fluorescence or bioluminescence energy transfer, the agonist [D-Ala^, D-Leu^]enkephalin had no effect on the homo­ oligomerisation status of the 6-opioid receptor and actually increased the hetero­ oligomerisation between the 6-opioid and p2-adrenergic receptors (McVey et al,

2001). Whether the oligomers demonstrated in co-immunoprecipitation and resonance energy experiments actually represents functionally relevant complexes, or simply receptors in close proximity to one another remains to be determined. There is some evidence for the localisation/clustering of GPCRs to lipid rafts, microdomains in the cell membrane that are enriched in cholesterol and sphingolipid (Anderson, 1998).