The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

The hydrophilic beta2a subunit of the L-type calcium channel was recently shown to be a membrane-localized, post-translationally modified protein (Chien, A. J., Zhao, X. L., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036-30044). In this study, we demonstrate that the rat beta2a subunit was palmitoylated through a hydroxylamine-sensitive thioester linkage. Palmitoylation required a pair of cysteines in the N terminus, Cys3 and Cys4; mutation of these residues to serines resulted in mutant beta2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient beta2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type beta2a. However, channels formed with a palmitoylation-deficient beta2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the beta2a subunit, they were important determinants of channel modulation by the beta2a subunit. Three other known beta subunits that were analyzed were not palmitoylated, suggesting that palmitoylation could provide a basis for the regulation of L-type channels through modification of a specific beta isoform.

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the ␤ 2 -adrenergic receptor (␤ 2 AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of ␤ 2 AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated ␤ 2 AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonistreceptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.Agonist binding activates G protein 1 -coupled receptors and initiates two intimately intertwined cascades of events, resulting in signal transduction and signal termination (desensitization). The receptor-agonist complex initially interacts with G protein(s) to form a transient agonist-receptor-G protein ternary complex that is the first intermediate in transmembrane signaling (1, 2). This ternary complex has a higher affinity for agonists than receptor alone (1, 2). Formation of this complex promotes GDP release from the G protein, which is followed by rapid GTP binding and dissociation of the active G␣⅐GTP and G␤␥ subunits. The agonist-occupied receptors are then phosphorylated by G protein-coupled receptor kinases, resulting in arrestin binding and consequent disruption of receptor-G protein interaction (3). Recent studies suggest that arrestin binding also targets the receptors for internalization (4, 5), apparently by virtue of the ability of non-visual arrestins to interact with clathrin (6), a process that appears to be a prerequisite for resensitization (3). Thus, the formation of the arrestin-receptor complex is not only the final step of signal termination but also an initial step of subsequent resensitization, representing a critical juncture in the signaling process. Because of this the arrestin-receptor complex appears to be a tempting target for a more detailed characterization. EXPERIMENTAL PROCEDURESArrestin Expression in Escherichia coli and Purification-Bovine arrestin cDNAs were subcloned using the NcoI and HindIII sites of pTrcB (Invitrogen). BL-21 cells transformed with the pTrcB-arrestin constructs were grown...

Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergic receptors (mAChR) from G-proteins and sequestration of the receptors away from the cell surface. However, mutant m2 mAChRs were identified that were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs. We have tested whether these properties might stem from differences in the abilities of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor/Gprotein uncoupling and internalization. We have determined that arrestin binding requires phosphorylation at a cluster of Ser/Thr residues in amino acids 307-311 in the m2 mAChR. A strong correlation was found between the ability of WT and mutant receptors to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays. However, the phosphorylation-dependent internalization of the m2 mAChR in HEK-tsA201 cells did not require arrestins and did not proceed via clathrin-mediated endocytosis. While the m2 mAChR was able to enter a clathrin-and arrestin-dependent pathway when arrestin 2 or arrestin 3 was significantly overexpressed, the preferred pathway of internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may involve arrestin-dependent and -independent events.