؊4 M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1 ⁄2 ‫؍‬ 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but downregulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loopindependent pathways.Loss of the cell's response to agonist acting at G proteincoupled receptors can occur in three phases: uncoupling from G proteins, sequestration/internalization, and down-regulation of the receptors. Many G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) 1 in an agonist-dependent manner as a major mechanism in receptor regulation (for review, see Refs. 1 and 2). Muscarinic acetylcholine receptor m2 subtypes have also been shown to be phosphorylated by GRK2 (␤-adrenergic receptor kinase 1) (3, 4) and other GRKs (5, 6). In addition, phosphorylation of ␤ 2 -adrenergic receptors by GRK2 may be involved in the uncoupling of ␤ 2 -adrenergic receptors from G protein G s (1, 2). The phosphorylated ␤ 2 -adrenergic receptors were reported to be uncoupled from G proteins because of their interaction with ␤-arrestin (1, 2, 7). Indeed, phosphorylation by GRKs has been reported to correlate with uncoupling for several G protein-coupled receptors including muscarinic m2 (8), ␣ 1B -adrenergic (9), ␣ 2 -adrenergic (10), thrombin (11), dopamine D1A (12), and thyrotropin receptors (13). ␤-Arrestin and arrestin 3 (␤-arrestin 2) were shown to interact with m2 muscarinic as well as with ␤ 2 -adrenergic receptors (14,15). Sequestration/internalization of ␤ 2 -adrenergic receptors seemed to be independent of phosphorylation by GRK2 on the basis of results with ␤ 2 -adrenergic receptor mutants lacking phosphorylation sites or GRK-specific inhibitors (16 -20). On the other hand, the agonist-induced sequestration of hm2 receptors expressed in HEK293 cells is hampered by deletion of the third intracellular loop (I3-loop) which includes the GRK2 phosphorylation sites (21,22). Moreover, agonist-dependent phosphorylation and sequestration of m2 receptors expressed in COS-7 cells are facilitated by coexpression of GRK2 and attenuated by coexpression of a dominant-negative mutant of GRK2 (DN-GRK2) that lacks kinase activity (23). Recently, Ferguson et al. have reexamined the relationship between the phosphorylation by GRK2 and sequestration of ␤ 2 -adrenergic receptors, demonstrating that phosphorylation by GRK2 (24) or other GRKs (25) facilitates sequestration of ␤ 2 -adrenergic receptors. Phosphorylation facilitates ␤-arrestin binding to ␤ 2 -adrenergic receptors (26) and thereby appears to enhance sequestration, possibly interacting with clathrin (27), a major * This work was supported in part by grants from the Japan Society for the Promotion of Science, the Ministry of Education, Science, and Culture of Japan, and the Japan Health Science Founda...

No abstract

The cardiac muscarinic receptor‐K+ channel system was reconstructed in Chinese hamster ovary (CHO) cells by transfecting the cells with the various components of the system. The activity of the muscarinic K+ channel was measured with the cell‐attached configuration of the patch clamp technique. In CHO cells transfected with the channel (Kir3.1/Kir3.4), receptor (hm2) and receptor kinase (GRK2), on exposure to agonist, there was a decline in channel activity as a result of desensitization, similar to that in atrial cells. Whereas the desensitization was almost abolished by not transfecting with the receptor kinase or by transfecting with a mutant receptor lacking phosphorylation sites, it was only reduced (by ≈39 %) by transfecting with a mutant receptor kinase with little kinase activity. These results suggest that the receptor kinase is responsible for desensitization of the muscarinic K+ channel and that this involves phosphorylation‐dependent and ‐independent mechanisms.

It is now well accepted that G protein-coupled receptors (GPCRs) can be directly associated, as either homo- or hetero-oligomers, to alter their functions. G protein-coupled purinergic receptors, classified as adenosine receptors, and P2Y receptors (ATP receptors) are also found to oligomerize each other to alter their pharmacology. Specifically, adenosine receptor of A1 subtype (A1R) is able to form a heteromeric complex with P2Y receptor of P2Y1 type (P2Y1R) either in heterologously transfected cells or in rat brain tissues, as demonstrated by coimmunoprecipitation or bioluminescence resonance energy transfer methods in addition to double immunocytochemistry. It is shown that the heteromerization between A1R and P2Y1R generates an adenosine receptor with P2Y-like agonistic pharmacology, i.e., a potent P2Y1R agonist, adenosine 5'-O-(2-thiodiphosphate), binds the A1R binding pocket of the A1R/P2Y1R complex and inhibits adenylyl cyclase activity via Gi/o protein. This hetero-oligomerization between adenosine receptor and P2Y receptor might be one of the mechanisms for the adenine nucleotide-mediated inhibition of neurotransmitter release. The oligomerization of purinergic receptors is thus considered as an important regulation system in the central nervous system.

Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.