Moritz Bünemann

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.

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.

REGULATORS OF G PROTEIN SIGNALING (RGS) PROTEINS CONSTITUTIVELY ACTIVATE GBETA GAMMA -GATED POTASSIUM CHANNELS

Here we report novel effects of regulators of G protein signaling (RGS) on G protein-regulated ion channels. RGS3 and RGS4 induced a substantial increase in currents through the Gβγ-regulated inwardly rectifying K+ channels,I K(ACh), in the absence of receptor activation. Concomitantly, the amount of current that could be activated by agonist was reduced. Pretreatment with pertussis toxin or a muscarinic receptor antagonist abolished agonist-induced currents but did not modify RGS effects. Cotransfection of cells with a Gβγ-binding protein significantly reduced the RGS4-induced basalI K(ACh) currents. The RGS proteins also modified the properties of another Gβγ effector, the N-type Ca2+ channels. These observations strongly suggest that RGS proteins increase the availability of Gβγ in addition to their previously described GTPase-activating function.

Role of the C terminus of the α1C(CaV1.2) Subunit in Membrane Targeting of Cardiac L-type Calcium Channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca2+) channel α1C (CaV1.2) and β subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the α1C subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the α1C subunit, a domain consisting of amino acid residues 1623–1666 (“targeting domain”) in the C terminus of the α1C subunit has been identified to be important for correct targeting of L-type Ca2+ channel complexes to the plasma membrane. Although cells expressing the wild-type α1C and β2a subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the α1C subunit that lack the targeting domain with the β2a subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the α1C subunit that were downstream of residues 1623–1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the α1C subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the α1C subunit was required for proper plasma membrane localization of the L-type Ca2+ channels.

Overexpressed A1 Adenosine Receptors Reduce Activation of Acetylcholine-Sensitive K+ Current by Native Muscarinic M2 Receptors in Rat Atrial Myocytes

In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K(+) current activated by a saturating concentration of adenosine (I(K(ACh),(Ado))) via A(1) receptors (A(1)Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (I(K(ACh),(ACh))) via muscarinic M(2) receptors. The half-time of activation of I(K(ACh),(Ado)) on a rapid exposure to agonist was approximately 4-fold longer than that of I(K(ACh),(ACh)). Furthermore, I(K(ACh),(Ado)) never showed fast desensitization. To study the importance of receptor density for A(1)R-I(K(ACh),(Ado)) signaling, adult atrial myocytes in vitro were transfected with cDNA encoding for rat brain A(1)R and enhanced green fluorescent protein (EGFP) as a reporter. Whole-cell current was measured on days 3 and 4 after transfection. Time-matched cells transfected with only the EGFP vector served as controls. In approximately 30% of EGFP-positive cells (group I), the density of I(K(ACh),(Ado)) was increased by 72%, and its half-time of activation was reduced. Density and kinetic properties of I(K(ACh),(ACh)) were not affected in this fraction. In approximately 70% of transfection-positive myocytes (group II), the density of I(K(ACh),(ACh)) was significantly reduced, its activation was slowed, and the fast desensitizing component was lost. Adenosine-induced currents were larger in group II than in group I, their activation rate was further increased, and a fast desensitizing component developed. These data indicate that in native myocytes the amplitude and activation kinetics of I(K(ACh),(Ado)) are limited by the expression of A(1)R. Overexpression of A(1)R negatively interferes with signal transduction via the muscarinic M(2) receptor-linked pathway, which might reflect a competition of receptors with a common pool of G proteins. Negative interference of an overexpressed receptor with physiological regulation of a target protein by a different receptor should be considered in attempts to use receptor overexpression for gene therapy.

In vivo downregulation of M2 receptors revealed by measurement of muscarinic K+ current in cultured guinea-pig atrial myocytes.

1 Muscarinic K+ current (IK(ACh)) elicited by acetylcholine (ACh) was measured in guinea‐pig atrial myocytes, which were either freshly isolated or cultured for up to 8 days. 2 The half‐time of activation of inward IK(ACh) by a saturating concentration (10 μm) of ACh decreased from ∼400 ms (in freshly isolated cells) to 250 ms after 6 days in culture. This was paralleled by an increase in the fast desensitizing component of IK(ACh). The density of steady‐state currents was not changed. Downregulation of M2 receptors by long‐term treatment of isolated myocytes with carbachol in vitro had opposite effects. 3 The EC50 of ACh for the activation of steady‐state −K(ACh) was reduced from 5 × 107 M (day 0) to 8 × 108 m (day 6). The shift in EC50 occurred with a half‐time of about 2 days, similar to the recovery from downregulation induced by treating atrial myocytes with carbachol in vitro. 4 The increase in sensitivity to ACh can be accounted for by an ∼6‐fold increase in the density of M2 receptors. 5 It is concluded that sensitization in culture reflects recovery from downregulation of M2 receptors due to the tonic vagal input to the heart in the intact animal.

Acidic Amino Acids Flanking Phosphorylation Sites in the M2 Muscarinic Receptor Regulate Receptor Phosphorylation, Internalization, and Interaction with Arrestins

The studies reported here address the molecular events underlying the interactions of arrestins with the M2 muscarinic acetylcholine receptor (mAChR). In particular, we focused on the role of receptor phosphorylation in this process. Agonist-dependent phosphorylation of the M2 mAChR can occur at clusters of serines and threonines at positions 286–290 (site P1) or 307–311 (site P2) in the third intracellular loop (Pals-Rylaarsdam, R., and Hosey, M. M. (1997)J. Biol. Chem. 272, 14152–14158). Phosphorylation at either P1 or P2 can support agonist-dependent internalization. However, phosphorylation at P2 is required for receptor interaction with arrestins (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682–26389). The present study investigated the role of acidic amino acids between P1 and P2 in regulating receptor phosphorylation, internalization, and receptor/arrestin interactions. Mutation of the acidic amino acids at positions 298–300 (site A1) and/or 304–305 (site A2) to alanines had significant effects on agonist-dependent phosphorylation. P2 was identified as the preferred site of agonist-dependent phosphorylation, and full phosphorylation at P2 required the acidic amino acids at A1 or their neutral counterparts. In contrast, phosphorylation at site P1 was dependent on site A2. In addition, sites A1 and A2 significantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestin2. Substitution of asparagine and glutamine for the aspartates and glutamates at sites A1 or A2 did not influence receptor phosphorylation but did influence arrestin interaction with the receptor. We propose that the amino acids at sites A1 and A2 play important roles in agonist-dependent phosphorylation at sites P2 and P1, respectively, and also play an important role in arrestin interactions with the M2 mAChR.

Molecular events associated with the regulation of signaling by M2 muscarinic receptors.

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.

Novel signalling events mediated by muscarinic receptor subtypes.

The M2 muscarinic acetylcholine receptor (mAChR) activates Gi protein coupled pathways, such as stimulation of G-protein activated inwardly rectifying K channels (GIRKs). Here we report a novel heterologous desensitization of these GIRK currents, which appeared to be specifically induced by M2/M4 mAChR stimulation, but not via adenosine (Ado) and alpha2-adrenergic receptors (AR). This heterologous desensitization reflected an inhibition of the GIRK signalling pathway downstream of G-protein activation. It was mediated in a membrane-delimited fashion via a PTX insensitive GTP dependent pathway and could be competed with exogenous Gbetagamma. The activation of M3 mAChR/Gq coupled receptors potently inhibited GIRK currents similar as M2 mAChR. By monitoring simultaneously the response of A1 adenosine receptor (AdoR) activation on N-type Ca2+ channels and GIRK channels, the stimulation of M3 mAChR was found to cause an inhibition of the Ado response in both effector systems, suggesting that the inhibition occurred at the level of the G-protein common to both effectors. These results indicated that Gq proteins inhibit pathways that are commonly regulated by Gbetagamma proteins.