M. Marlene Hosey

cAMP-Dependent Regulation of Cardiac L-Type Ca2+ Channels Requires Membrane Targeting of PKA and Phosphorylation of Channel Subunits

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.

Calcium channels: Molecular pharmacology, structure and regulation

It is well recognized that calcium (Ca) is an important regulatory element for many cellular processes. In most eukaryotic cells a diverse array of Ca transporting systems functions to maintain the steep concentration gradient between extracellular Ca, estimated to be in the millimolar range, and intracellular Ca, which can vary between 0.1-10 /xM, depending on the state of the cell. Specific and distinct pathways exist by which Ca enters and exits cells. Several different types of Ca transport systems serve to maintain the low concentration of intracellular Ca, by transporting Ca either out of the cell or into intracellular storage sites (for review, see Carafoli, 1987). These exi t pathways have been extensively studied, and until recently our knowledge of these systems was far greater than for pathways involved in Ca entry . The major entry pathway for Ca in many cell types is via plasma membrane Ca channels. Considerable progress has now been made in elucidating the properties of certain Ca channels. The purpose of this review is to summarize our current understanding of the molecular properties of Ca channels; particular emphasis will be given to the pharmacological and biochemical characterization of Ca channels, as well as the mechanisms by which they are regulated by neurotransmitter-mediated processes.

G‐protein coupled receptor kinases as modulators of G‐protein signalling

G‐protein coupled receptors (GPCRs) comprise one of the largest classes of signalling molecules. A wide diversity of activating ligands induce the active conformation of GPCRs and lead to signalling via heterotrimeric G‐proteins and downstream effectors. In addition, a complex series of reactions participate in the ‘turn‐off’ of GPCRs in both physiological and pharmacological settings. Some key players in the inactivation or ‘desensitization’ of GPCRs have been identified, whereas others remain the target of ongoing studies. G‐protein coupled receptor kinases (GRKs) specifically phosphorylate activated GPCRs and initiate homologous desensitization. Uncoupling proteins, such as members of the arrestin family, bind to the phosphorylated and activated GPCRs and cause desensitization by precluding further interactions of the GPCRs and G‐proteins. Adaptor proteins, including arrestins, and endocytic machinery participate in the internalization of GPCRs away from their normal signalling milieu. In this review we discuss the roles of these regulatory molecules as modulators of GPCR signalling.

Identification and Subcellular Localization of the Subunits of L-type Calcium Channels and Adenylyl Cyclase in Cardiac Myocytes

The properties of cardiac L-type channels have been well characterized electrophysiologically, and many such studies have demonstrated that the channels are regulated by a cAMP-dependent pathway. However, the subunit composition of native cardiac L-type calcium channels has not been completely defined. Furthermore, a very important question exists regarding the status of the C-terminal domain of the pore-forming α1subunit, as this domain has the potential to be the target of protein kinases but may be truncated as a result of post-translational processing. In the present studies, the α1C and β2 subunits were identified by subunit-specific antibodies after partial purification from heart membranes, or immunoprecipitation from cardiac myocytes. Both the β2and the full-length α1C subunits were found to be expressed and co-localized in intact cardiac myocytes along T-tubule membranes. Using a quantitative antibody binding analysis, we demonstrated that the majority of the α1C subunits in intact cardiac myocytes appear to be full-length. In addition, we observed that adenylyl cyclase is localized in a pattern similar to the channel subunits in cardiac myocytes. Taken together, our results provide new insights into the structural basis for understanding the regulation of L-type calcium channels by a cAMP-mediated signaling pathway.

Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit.

Activation of protein kinase A (PKA) through the β-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the β2 subunit are of functional significance, we coexpressed wild-type (WT) or mutant β2 subunits in tsA-201 cells together with an α1C subunit, α1CΔ1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated α1C subunit was similar to the truncated α1C subunit isolated from cardiac tissue not only in size (∼190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT β2 subunit, voltage-activated Ba2+ currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the β2 subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca2+currents may be at least partially caused by phosphorylation of the β2 subunit at Ser-478 and Ser-479.

Agonist-Receptor-Arrestin, an Alternative Ternary Complex with High Agonist Affinity

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 β2-adrenergic receptor (β2AR), 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 β2AR-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 β2AR 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 agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.

Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms.

The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphorylation, loss of high affinity agonist binding, receptor:G-protein uncoupling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contributions to the loss of signaling remain correlative. In addition, the nature of the kinases and the receptor domains which are involved in modulation of activity have only begun to be investigated. In order to directly measure the role of G-protein-coupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2 reduced agonist-dependent phosphorylation of the m2 mAChR by 50% and prevented acute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity. In contrast, the agonist-induced internalization of the m2 mAChR was unaffected by the GRK2 construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of the third intracellular loop were made which created m2 mAChRs that did not become phosphorylated in an agonist-dependent manner and did not desensitize. However, the mutant mAChRs retained the ability to internalize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; the likely sites of in vivo phosphorylation are in the central portion of the third intracellular loop (amino acids 282-323). These results also indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK phosphorylation of the receptor.

ARRESTIN-INDEPENDENT INTERNALIZATION OF THE M1, M3, AND M4 SUBTYPES OF MUSCARINIC CHOLINERGIC RECEPTORS

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682–23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the β2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.

Two Homologous Phosphorylation Domains Differentially Contribute to Desensitization and Internalization of the m2 Muscarinic Acetylcholine Receptor

Short term exposure of m2 muscarinic acetylcholine receptors (m2 mAChRs) to agonist causes a rapid phosphorylation of the activated receptors, followed by a profound loss in the ability of the m2 mAChR to activate its signaling pathways. We have used site-directed mutagenesis to identify two clusters of Ser/Thr residues in the third intracellular loop of the m2 mAChR that can serve as redundant targets for agonist-dependent phosphorylation. Mutation of both clusters of Ser/Thr residues to alanines abolished agonist-dependent phosphorylation, while wild-type levels of m2 mAChR phosphorylation were observed in mutant receptors with only one or the other cluster mutated. However, the functional effects of phosphorylation of these two “redundant” clusters were not equivalent. No receptor desensitization was observed in an m2 mAChR with residues Thr307–Ser311 mutated to alanine residues. In contrast, mutation of the other Ser/Thr cluster, residues Ser286–Ser290, to alanines produced a receptor that continued to desensitize. Internalization of the m2 mAChR was promoted by phosphorylation of either cluster, suggesting that distinct mechanisms with unique structural requirements act downstream of m2 mAChR phosphorylation to mediate receptor desensitization and receptor internalization.

Sorcin Associates with the Pore-forming Subunit of Voltage-dependent L-type Ca2+ Channels

Intracellular Ca2+release in muscle is governed by functional communication between the voltage-dependent L-type Ca2+ channel and the intracellular Ca2+ release channel by processes that are incompletely understood. We previously showed that sorcin binds to cardiac Ca2+ release channel/ryanodine receptors and decreases channel open probability in planar lipid bilayers. In addition, we showed that sorcin antibody immunoprecipitates ryanodine receptors from metabolically labeled cardiac myocytes along with a second protein having a molecular weight similar to that of the α1 subunit of cardiac L-type Ca2+ channels. We now demonstrate that sorcin biochemically associates with cardiac and skeletal muscle L-type Ca2+ channels specifically within the cytoplasmically oriented C-terminal region of the α1 subunits, providing evidence that the second protein recovered by sorcin antibody from cardiac myocytes was the 240-kDa L-type Ca2+ channel α1 subunit. Anti-sorcin antibody immunoprecipitated full-length α1 subunits from cardiac myocytes, C2C12 myotubes, and transfected non-muscle cells expressing α1 subunits. In contrast, the anti-sorcin antibody did not immunoprecipitate C-terminal truncated forms of α1 subunits that were detected in myotubes. Recombinant sorcin bound to cardiac and skeletal HIS6-tagged α1 C termini immobilized on Ni2+ resin. Additionally, anti-sorcin antibody immunoprecipitated C-terminal fragments of the cardiac α1 subunit exogenously expressed in mammalian cells. The results identified a putative sorcin binding domain within the C terminus of the α1 subunit. These observations, along with the demonstration that sorcin accumulated substantially during physiological maturation of the excitation-contraction coupling apparatus in developing postnatal rat heart and differentiating C2C12 muscle cells, suggest that sorcin may mediate interchannel communication during excitation-contraction coupling in heart and skeletal muscle.