The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the ␥ subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzymereceptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.
G protein-coupled receptor kinases (GRKs)1 form a family of serine/threonine protein kinases that phosphorylate specifically agonist-bound or -activated G protein-coupled receptors (1). A common feature of this family is multi-site phosphorylation of receptor substrates in response to agonist occupancy (2). GRK-mediated receptor phosphorylation promotes the binding of arrestin, thereby uncoupling the receptor from G proteins and terminating receptor signaling (1). Based on sequence and functional similarities, the GRK family has been divided into three subfamilies: the rhodopsin kinase subfamily (RK or GRK1), the -adrenergic receptor kinase subfamily (GRK2 and GRK3), and the GRK4 subfamily (GRK4, GRK5, and GRK6). Recently, a splice variant of human GRK1 (termed GRK1b) has been identified (3), and a candidate cone opsin kinase (termed GRK7) has been cloned from cone-and roddominant mammalian retinas (4).GRKs share a similar structural organization: a centrally located catalytic domain, an N-terminal domain, and a C-terminal domain. The N-terminal domains of GRKs, which are similar in size (ϳ185 amino acids), is believed to be responsible for receptor recognition. The C-terminal domains, which share a fair degree of structural homology but vary in length, can be divided further into two subdomains: a relatively conserved region and a variable region (1, 2). The variable region in the C-terminal domain of GRKs contains the site of several posttranslational modifications and regulatory protein-protein interactions, which appear to be involved in the membrane and receptor targeting of GRKs (1, 2). Differe...