Oliver Kudlacek

Binding of Calmodulin to the D2-Dopamine Receptor Reduces Receptor Signaling by Arresting the G Protein Activation Switch

Signaling by D2-dopamine receptors in neurons likely proceeds in the presence of Ca2+ oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca2+ and the D2 receptor. By activation of calmodulin (CaM), Ca2+ directly inhibits the D2 receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH2-terminal end of the third loop, a domain involved in activating Gi/o. A peptide fragment encompassing this domain (D2N) bound dansylated CaM in a Ca2+-dependent manner (KD ∼ 0.1 μm). (ii) Activation of purified Gαi1 by D2N, and D2receptor-promoted GTPγS (guanosine 5′-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca2+/CaM (IC50 ∼ 0.1 μm). (iii) If Ca2+ influx was elicited in D2 receptor-expressing HEK293 cells, agonist-dependent inhibition of cAMP formation decreased. This effect was not seen with other Gi-coupled receptors (A1-adenosine and Mel1A-melatonin receptor). (iv) The D2 receptor was retained by immobilized CaM and radiolabeled CaM was co-immunoprecipitated with the receptor. Specifically, inhibition by CaM does not result from uncoupling the D2 receptor from its cognate G protein(s); rather, CaM directly targets the D2 receptor to block the receptor-operated G protein activation switch.

The N Terminus of Monoamine Transporters Is a Lever Required for the Action of Amphetamines

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.

MAP Kinase Stimulation by cAMP Does Not Require RAP1 but SRC Family Kinases

The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A2A-adenosine receptor, a prototypical Gs-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A2A cells). In CHO-A2A cells, the stimulation of the A2A-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A2A-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A2A-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A2A-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A2A-receptor- and the forskolin-induced MAP kinase stimulation (IC50 = 50 nm); this was also seen in PC12 cells, which express the A2A-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the β2-adrenergic receptor, is apparently contingent on receptor endocytosis.

Two Discontinuous Segments in the Carboxyl Terminus Are Required for Membrane Targeting of the Rat γ-Aminobutyric Acid Transporter-1 (GAT1)

Like all members of the Na+/Cl--dependent neurotransmitter transporter family, the rat γ-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Δ37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Δ27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Δ27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Δ3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Δ37 and GAT1-Δ32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif (569VMI571) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.

The Serotonin Transporter Is an Exclusive Client of the Coat Protein Complex II (COPII) Component SEC24C

The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif (607RI608) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.

Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters

Highlights • We quantified adulterants in street drugs sold as cocaine.• We analyzed effects of the most common adulterant levamisole, on neurotransmitter transporters.• Differences in the selectivity of levamisole can be explained by homology modelling and docking.• Aminorex, a metabolite of levamisole, modulates neurotransmitter transporters directly.• Depending on the transporter, aminorex acts as a blocker or as a releaser.

Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells

The adenosine A2A receptor and the dopamine D2 receptor are prototypically coupled to Gs and Gi/Go, respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor–G protein coupling. We tested this model in PC12 cells which endogenously express A2A receptors. The human D2 receptor was introduced into PC12 cells by stable transfection. A2A-agonist-mediated inhibition of D2 agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D2 agonist quinpirole depended on the expression level of the D2 receptor: at low and high receptor levels, the A2A-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A2A-receptor-mediated cAMP formation was inhibited by other Gi/Go-coupled receptors that were either endogenously present (P2y12-like receptor for ADP) or stably expressed after transfection (A1 adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca2+ channels were inhibited by the endogenous P2Y receptor and by the heterologously expressed A1 receptor but not by the D2 receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D2 and A2A receptors are closely associated, but they highlight the fact that this interaction can also support synergism.

Mutations in the carboxyl-terminal SEC24 binding motif of the serotonin transporter impair folding of the transporter.

The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG601,602, RII607–609) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI607,608 is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG601,602 and RI607,608 was impaired folding, because mutated transporters failed to bind the inhibitor [3H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [3H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI607,608AA and SERT-RII607–609AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter IMPLICATIONS FOR ANGELMAN SYNDROME

Background: αCaMKII modulates amphetamine-induced dopamine transporter-mediated substrate efflux. Results: Mice with ablated or blunted αCaMKII function show decreased amphetamine-triggered efflux. Conclusion: Dopamine transporter function is impaired in mice with targeted αCaMKII mutations and in a mouse model of the Angelman syndrome. Significance: Such new insights into dopamine transporter function may further illuminate the complex pathophysiology of the Angelman syndrome. The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP+) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKIIT305D), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP+ efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.