cAMP guided his way: a life for G protein-mediated signal transduction and molecular pharmacology-tribute to Karl H. Jakobs.

Karl H. Jakobs, former editor-in-chief of Naunyn-Schmiedeberg's Archives of Pharmacology and renowned molecular pharmacologist, passed away in April 2018. In this article, his scientific achievements regarding G protein-mediated signal transduction and regulation of canonical pathways are summarized. Particularly, the discovery of inhibitory G proteins for adenylyl cyclase, methods for the analysis of receptor-G protein interactions, GTP supply by nucleoside diphosphate kinases, mechanisms in phospholipase C and phospholipase D activity regulation, as well as the development of the concept of sphingosine-1-phosphate as extra- and intracellular messenger will presented. His seminal scientific and methodological contributions are put in a general and timely perspective to display and honor his outstanding input to the current knowledge in molecular pharmacology.

Homo- and heteromeric assembly of TRPV channel subunits.

The vanilloid receptor-related TRP channels (TRPV1-6) mediate thermosensation, pain perception and epithelial Ca(2+) entry. As the specificity of TRPV channel heteromerization and determinants governing the assembly of TRPV subunits were largely elusive, we investigated the TRPV homo- and heteromultimerization. To analyze the assembly of TRPV subunits in living cells, we generated fluorescent fusion proteins or FLAG-tagged TRPV channel subunits. The interaction between TRPV subunits was assessed by analysis of the subcellular colocalization, fluorescence resonance energy transfer and coimmunoprecipitation. Our results demonstrate that TRPV channel subunits do not combine arbitrarily. With the exception of TRPV5 and TRPV6, TRPV channel subunits preferentially assemble into homomeric complexes. Truncation of TRPV1, expression of cytosolic termini of TRPV1 or TRPV4 and construction of chimeric TRPV channel subunits revealed that the specificity and the affinity of the subunit interaction is synergistically provided by interaction modules located in the transmembrane domains and in the cytosolic termini. The relative contribution of intramolecularly linked interaction modules presumably controls the overall affinity and the specificity of TRPV channel assembly.

Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected].

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.

TRPV1 acts as proton channel to induce acidification in nociceptive neurons.

The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca(2+)-dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na(+), Mg(2+), and Ca(2+). The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia.

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H2O2), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1-5 mM H2O2 for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H2O2 and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H2O2 in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia.

FRET-based analysis of TRPC subunit stoichiometry.

By analogy to other cation channel subunits with six transmembrane-spanning domains, the seven members of the "classical" or "canonical" transient receptor potential channels (TRPC) family are believed to assemble into homo- or heterotetrameric complexes. These complexes have been verified by classical methods such as coimmunoprecipitation, crosslinking analysis or functional assays applying dominant negative pore mutants. More recently, fluorescence resonance energy transfer (FRET)-a measure for the close proximity of fluorescent molecules-has become instrumental in monitoring protein assembly in living cells. Here we demonstrate further possibilities and verification procedures of the FRET technology to test the assembly of ion channel subunits. Temporally and spatially resolved FRET imaging demonstrates an early assembly of TRPC subunits in the endoplasmic reticulum and the Golgi apparatus. Confocal FRET imaging verifies FRET signals over the plasma membrane at high spatial resolution. Taking advantage of the quantitative analysis of digital video imaging, we demonstrate that FRET between TRPC subunits is only poorly concentration-dependent. Moreover, a correlation between the efficiency of energy transfer and the molar ratio of the FRET donor to the acceptor was exploited to verify the tetrameric stoichiometry of TRPC complexes. Finally, we introduce a competition-FRET assay to test the ability of wild-type TRPC subunits to recruit fluorescent TRPC subunits into separate channel complexes.

Receptor-dependent RhoA activation in G12/G13-deficient cells: genetic evidence for an involvement of Gq/G11.

The small GTPase RhoA is involved in the regulation of various cellular functions like the remodeling of the actin cytoskeleton and the induction of transcriptional activity. G-protein-coupled receptors (GPCRs), which are able to activate Gq/G11 and G12/G13 are major upstream regulators of RhoA activity, and G12/G13 have been shown to couple GPCRs to the activation of Rho by regulating the activity of a subfamily of RhoGEF proteins. However, the possible contribution of Gq/G11 to the regulation of RhoA activity via GPCRs is controversial. We have used a genetic approach to study the role of heterotrimeric G-proteins in the activation of RhoA via endogenous GPCRs. In pertussis toxin-treated Galpha12/Galpha13-deficient as well as in Galphaq/Galpha11-deficient mouse embryonic fibroblasts (MEFs), in which coupling of receptors is restricted to Gq/G11 and G12/G13, respectively, receptor activation results in Rho activation. Rho activation induced by receptor agonists via Gq/G11 occurs with lower potency than Rho activation via G12/G13. Activation of RhoA via Gq/G11 is not affected by the phospholipase-C blocker U73122 or the Ca2+-chelator BAPTA, but can be blocked by a dominant-negative mutant of the RhoGEF protein LARG. Our data clearly show that G12/G13 as well as Gq/G11 alone can couple GPCRs to the rapid activation of RhoA. Gq/G11-mediated RhoA activation occurs independently of phospholipase C-beta and appears to involve LARG.

Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site.

Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.

Molecular and functional characterization of the melastatin-related cation channel TRPM3.

Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca2+ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca2+ and Mn2+ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios PCa/PNa of 1.57 and PNa/PCs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mm Cs+, 140 mm Na+, or 100 mm Ca2+ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca2+ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca2+ homeostasis.

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma.

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.

Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth.

Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). Unlike most other TRP-related channels, which are inhibited by La(3+) and Gd(3+), currents through TRPC4 and TRPC5 are potentiated by La(3+). Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La(3+) or Gd(3+), whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La(3+) or Gd(3+) at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La(3+), but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu(543) and Glu(595)/Glu(598), situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu(595)/Glu(598) in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La(3+) and Gd(3+) have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La(3+) at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.

Aminoglycoside pretreatment partially restores the function of truncated V(2) vasopressin receptors found in patients with nephrogenic diabetes insipidus.

By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V(2) vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.

Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells.

The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms. Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding. We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs. Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKC alpha accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKC epsilon was delayed and attenuated. In BAPTA-loaded cells, however, a selective translocation of PKC epsilon, but not of coexpressed PKC alpha, was evident. A membrane-permeable DAG analogue displayed a higher binding affinity for PKC epsilon than for PKC alpha. Subsequent photolysis of caged Ca2+ immediately recruited PKC alpha to the membrane, and DAG-bound PKC epsilon was displaced. At low expression levels of PKC epsilon, PKC alpha concentration dependently prevented the PKC epsilon translocation with half-maximal effects at equimolar coexpression. Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels. We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation.

Structural requirements for mutational lutropin/choriogonadotropin receptor activation.

Different activation mechanisms of glycoprotein hormone receptors, which are members of the G protein-coupled receptor superfamily, have been proposed. For example, the large ectodomain of glycoprotein hormone receptors may function as an inverse agonist keeping the transmembrane domain in an inactive conformation. To provide support for this hypothesis, we have generated different lutropin/choriogonadotropin receptor (LHR) constructs lacking the ectodomain. Although some ectodomain-deficient LHR constructs were targeted to the cell surface, cAMP levels remained unchanged under basal conditions and agonist application but could be increased by a mutation within the transmembrane domain 6 (D578H). Taking advantage of a constitutive activating mutation (S277N) located in the extracellular domain, we showed that the intact leucine-rich repeat-containing ectodomain is essential for constitutive activation of the LHR by mutation of the hinge region. Our findings support an activation scenario in which agonist binding or mutational alterations expose a structure within the ectodomain, which then activates the transmembrane core.

The PDZ scaffold protein INAD abolishes apparent store-dependent regulation of the light-activated cation channel TRP.

In fly photoreceptor cells, light initiates a G protein-coupled phospholipase Cb-dependent signaling cascade that results in the depolarization of the cell membrane, which is mediated by the cation channels TRP and TRPL. Together with phospholipase Cb and an eye-specific protein kinase C, TRP is tethered to the scaffolding protein INAD, which forms a multimolecular signaling complex. Divergent data from expressed TRP and studies from photoreceptor cells have brought up a controversy whether or not a capacitative calcium entry (CCE) mechanism is involved in the Drosophila phototransduction pathway. Our initial characterization of TRP from photoreceptors of Calliphora vicina supported the hypothesis of a CCE mechanism, as heterologously expressed TRP was stimulated after application of thapsigargin. The situation changed when the PDZ domain protein INAD was coexpressed with TRP. In cells coexpressing TRP and INAD, no calcium entry was detectable on application of store depletion protocols. Suppression of CCE by INAD was not observed when the described interaction was disrupted by mutations in TRP and INAD. Our data show that apparent activation of TRP by CCE is abolished by INAD. Within the complex, the proteins necessary for phototransduction mutually influence their activities. The results support the hypothesis of a store-independent activation of TRP.

Subunit composition of mammalian transient receptor potential channels in living cells.

Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches--i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation--we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies--e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells.

G-protein betagamma-subunits contribute to the coupling specificity of the beta2-adrenergic receptor to G(s).

Receptors and heterotrimeric G-proteins interact with a high degree of specificity, the molecular basis of which is only partially understood. In the present study, we analyzed the influence of different G-protein betagamma-subunits on the coupling of the beta2-adrenergic receptor to G(s). Sf9-cells were infected with baculoviruses coding for the beta2-adrenergic receptor, alpha(s,Short) or alpha(s,Long), and various beta- and gamma-subunits. The ability of different beta- and gamma-subunits to correctly dimerize was assessed by limited proteolysis of proteins expressed in Sf9-cells and additionally by analysis of beta/gamma-interaction in the yeast two-hybrid system. Agonist-induced GTPgammaS-binding to alpha(s,Short)beta(1)gamma-trimers was significantly higher than to alpha(s,Short)beta2gamma-combinations, when gamma4, gamma5, or gamma7 were co-expressed. Because beta(5) did not support coupling of the beta(2)-adrenergic receptor to G(s), the 87 C-terminal amino acids of Gbeta(5) assumed to encompass the beta-subunit interface with the receptor were substituted by the corresponding sequence of beta(1). Whereas this beta(5)/beta(1)-chimera did not promote GTPgammaS-binding to alpha(s), histamine H(1)-receptor-dependent GTPgammaS-binding to alpha(q) was supported by this chimeric beta-subunit and by wild-type beta(5). Our findings argue that the betagamma-subunit composition contributes directly to the specificity of beta(2)-adrenergic receptor-mediated G(s)-activation.

TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells.

To investigate the possible role of members of the mammalian transient receptor potential (TRP) channel family (TRPC1-7) in vasoconstrictor-induced Ca(2+) entry in vascular smooth muscle cells, we studied [Arg(8)]-vasopressin (AVP)-activated channels in A7r5 aortic smooth muscle cells. AVP induced an increase in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) consisting of Ca(2+) release and Ca(2+) influx. Whole cell recordings revealed the activation of a nonselective cation current with a doubly rectifying current-voltage relation strikingly similar to those described for some heterologously expressed TRPC isoforms. The current was also stimulated by direct activation of G proteins as well as by activation of the phospholipase Cgamma-coupled platelet-derived growth factor receptor. Currents were not activated by store depletion or increased [Ca(2+)](i). Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property of the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents in A7r5 cells were increased by flufenamate. Northern hybridization revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that TRPC6 is a molecular component of receptor-stimulated Ca(2+)-permeable cation channels in A7r5 smooth muscle cells.