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SUMMARYSelective Na+-dependent re-uptake of biogenic monoamines at mammalian nerve synapses is accomplished by three types of solute-linked carrier family 6 (SLC6) membrane transporter with high affinity for serotonin(SERTs), dopamine (DATs) and norepinephrine (NETs). An additional SLC6 monoamine transporter (OAT), is responsible for the selective uptake of the phenolamines octopamine and tyramine by insect neurons. We have characterized a similar high-affinity phenoloamine transporter expressed in the CNS of the earthworm Lumbricus terrestris. Phylogenetic analysis of its protein sequence clusters it with both arthropod phenolamine and chordate catecholamine transporters. To clarify the relationships among metazoan monoamine transporters we identified representatives in the major branches of metazoan evolution by polymerase chain reaction (PCR)-amplifying conserved cDNA fragments from isolated nervous tissue and by analyzing available genomic data. Analysis of conserved motifs in the sequence data suggest that the presumed common ancestor of modern-day Bilateria expressed at least three functionally distinct monoamine transporters in its nervous system: a SERT currently found throughout bilaterian phyla, a DAT now restricted in distribution to protostome invertebrates and echinoderms and a third monoamine transporter (MAT), widely represented in contemporary Bilateria, that is selective for catecholamines and/or phenolamines. Chordate DATs, NETs,epinephrine transporters (ETs) and arthropod and annelid OATs all belong to the MAT clade. Contemporary invertebrate and chordate DATs belong to different SLC6 clades. Furthermore, the genes for dopamine and norepinephrine transporters of vertebrates are paralogous, apparently having arisen through duplication of an invertebrate MAT gene after the loss of an invertebrate-type DAT gene in a basal protochordate.

The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for Gialpha and Goalphaand a guanine nucleotide dissociation inhibitor (GDI) for Gialpha. This GDI activity is isolated to a region of the protein distinct from the RGS domain that contains an additional G protein-binding domain (RBD/GL). Here, we report that RGS14 missing its RGS domain (R14-RBD/GL) binds directly to Go and Gi to modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. In brain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14) bind Goalpha-GDP, Gialpha-GDP, and also Gbetagamma. When reconstituted with M2 muscarinic receptors (M2) plus either Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulates the steady-state GTPase activities of both G proteins, whereas R14-RBD/GL has little or no effect. R14-RBD/GL potentiates RGS4 GAP activity in membrane-based assays by increasing the apparent affinity of RGS4 for Gialpha and Goalpha, suggesting a cooperative interaction between the RBD/GL domain, RGS4, and Galpha. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assays with purified Gialpha or Goalpha, suggesting that membranes and/or receptors are required for this activity. When these findings are taken together, they indicate that regions of RGS14 outside of the RGS domain can bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Galphanucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDI activity.

RGS2 and RGS4 were studied for their effects as GTPase activating proteins (GAPs) on receptor-activated Gi in a novel steady-state assay using membranes from Sf9 cells quadruply infected with baculoviruses encoding the m2 muscarinic receptor, Gαi2, Gβ1, and Gγ2. In the presence of the muscarinic agonist carbachol, regulator of G protein signaling 2 (RGS2) and RGS4 each produced up to a 10-fold increase in agonist-dependent GTPase activity. The observed K m for GTP in this system was increased in the presence of RGS4. NaCl and KCl inhibited the GAP activities of both RGS2 and RGS4, although they had no effect on GTPase activity in the absence of RGS proteins. MgCl2 had a complex effect on GTPase activity, with optimal RGS2 and RGS4 GAP activities occurring, respectively, at high micromolar and low millimolar concentrations of free Mg2+. The concentration dependence of RGS GAP activity was assessed, and RGS4 was found to be more potent than RGS2 by up to an order of magnitude. This direct observation confirms a similar difference in potency found when these two RGS proteins were compared for their ability to inhibit signaling downstream of Gi([Heximer et al., 1999][1]). RGS2 yielded Hill coefficients greater than 2.0, suggesting that it may bind in a positively cooperative manner to oligomeric structures containing more than one G protein. Furthermore, RGS4 yielded a bell-shaped dose-dependence under low magnesium (0.5 mM) conditions, which is also consistent with the idea of RGS cooperativity. [1]: #ref-16

Heterotrimeric G proteins convey receptor signals to intracellular effectors. Superimposed over the basic GPCR-G protein-effector scheme are three types of auxiliary proteins that also modulate Gα. Regulator of G protein signaling proteins and G protein signaling modifier proteins respectively promote GTPase activity and hinder GTP/GDP exchange to limit Gα activation. There are also diverse proteins that, like GPCRs, can promote nucleotide exchange and thus activation. Here we review the impact of these auxiliary proteins on GPCR signaling. Although their precise physiological functions are not yet clear, all of them can produce significant effects in experimental systems. These signaling changes are generally consistent with established effects on isolated Gα; however, the activation state of Gα is seldom verified and many such changes appear also to reflect the physical disruption of or indirect effects on interactions between Gα and its associated GPCR, Gβγ, and/or effector.

Heterotrimeric G proteins convey receptor signals to intracellular effectors. Superimposed over the basic GPCR–G protein–effector scheme are three types of auxiliary proteins that also modulate Gα. Regulator of G protein signaling proteins and G protein signaling modifier proteins respectively promote GTPase activity and hinder GTP/GDP exchange to limit Gα activation. There are also diverse proteins that, like GPCRs, can promote nucleotide exchange and thus activation. Here we review the impact of these auxiliary proteins on GPCR signaling. Although their precise physiological functions are not yet clear, all of them can produce significant effects in experimental systems. These signaling changes are generally consistent with established effects on isolated Gα; however, the activation state of Gα is seldom verified and many such changes appear also to reflect the physical disruption of or indirect effects on interactions between Gα and its associated GPCR, Gβγ, and/or effector.

We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (K(m) and K(i) range, 0.4 microM-2.7 microM) and capacity ((3)H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity ((3)H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.