Probing the roles of protein kinases in g-protein-coupled receptor desensitization.

For decades we have understood that G-protein-coupled receptors (GPCRs) are crucial links in the relay of information from extracellular stimuli to intracellular responses. Their importance is highlighted by the wide variety of GPCRs encoded in the human genome (Fredriksson et al., 2003; Perez, 2003) and the multiple mechanisms by which they are regulated (Clark, 1986; Palczewski and Benovic, 1991; Kohout and Lefkowitz, 2003). The potential for pharmacological intervention in GPCR function has led to an enormous effort to determine the mechanisms of GPCR activation and desensitization after stimulation by hormones and drugs. In this issue of Molecular Pharmacology, Willets et al. (2003) investigate the mechanisms of M3 muscarinic acetylcholine receptor (mAChR) desensitization in intact cells after agonist stimulation. These authors have confronted a number of the problems associated with the identification of protein kinases that mediate desensitization of GPCRs (see Table 1). It is refreshing to read such a considered discussion of the types of problems that have hindered efforts to understand protein kinase regulation of GPCRs. Perhaps the most well understood protein kinase-meditated desensitization of a GPCR is that of rhodopsin. The large concentrations of rhodopsin present in rod outer segments ( 3 mM) have allowed detailed analysis of its activation, regulation, and structure (Molday, 1998; Filipek et al., 2003) and led to the early recognition of the roles of GRKs and arrestin in GPCR desensitization. Studies of the rod outer segment have also led the way in understanding the regulation of GRKs by recoverin (Polans et al., 1996), and the role of RGS proteins in the desensitization process (Wensel, 2002). However, despite the ease of analysis of this system, it has not been immune to the problems listed in Table 1. For example, early studies based upon cell-free preparations with purified components, not intact rod outer segments, demonstrated that seven or eight residues in the carboxyl terminal tail were phosphorylated by rhodopsin kinase (Wilden and Kuhn, 1982). More recently, Palczewski and colleagues (Ohguro et al., 1995; Maeda et al., 2003) have shown that, in vivo, only one residue is initially phosphorylated by rhodopsin kinase and that with more prolonged light stimulation, two additional residues are phosphorylated. The elegant body of work on rhodopsin desensitization illustrates how crucial the synthesis of in vitro and in vivo approaches is to the illumination of complex mechanisms of regulation, and it has guided the study of protein kinase regulation of GPCRs in a variety of cellular systems. An example of that has been the study of the role of protein kinases in the desensitization of the 2-adrenergic receptor (Clark, 1986; Palczewski and Benovic, 1991; Premont et al., 1995; Kohout and Lefkowitz, 2003). All of the problems outlined in Table 1 have been encountered in studies of the role of 2-adrenergic receptor kinase (GRK2) in desensitization. As with the rod outer segment system, it has taken the merger of in vitro work that was crucial to the discovery of GRK2 with in vivo studies to approach a consensus. To illustrate this point, it was initially thought that as many as six to nine sites were phosphorylated by GRKs on the distal C-tail of the receptor based on cell-free studies with purified components (Kim et al., 1993; Fredericks et al., 1996). However, more recent evidence from studies of intact cells indicates that the number of sites affecting desensitization is considerably less and that their location is more proximal on the C-tail than originally thought (Seibold et al., 2000; Friedman et al., 2003). In the work by Willets et al. (2003), it is noted that cell-free studies identified GRK2 and GRK3 as the protein kinases that phosphorylate the M3 mAChR (DebBurman et al., 1995) whereas GRK6 was ineffective. Using dominant negative GRK2, GRK3, and GRK6, Willets et al. (2003) now show that muscarinic agonist-induced phosphorylation of endogenous