The armadillo protein SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), such as Rac1 and RhoA. Because the PBR resembles a nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has significant NLS activity when it is fused to green fluorescent protein (GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR has very poor NLS activity when it is fused to GFP or in the context of full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR. Conversely, SmgGDS nuclear accumulation is diminished by interactions with RhoA. An SmgGDS nuclear export signal sequence that we identified promotes SmgGDS nuclear export. These results suggest that SmgGDS⅐ Rac1 complexes accumulate in the nucleus because the Rac1 PBR has NLS activity and because Rac1 supplies the appropriate GTP-dependent signal. In contrast, SmgGDS⅐RhoA complexes accumulate in the cytoplasm because the RhoA PBR does not have NLS activity. This model may be applicable to other armadillo proteins in addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA also provide stimulatory and inhibitory signals, respectively, for the nuclear accumulation of p120 catenin. These results indicate that small GTPases with a PBR can regulate the nuclear transport of armadillo proteins.Armadillo (ARM) 1 family proteins that contain multiple copies of the ϳ42-amino acid (aa) ARM motif include SmgGDS, p120 catenin (p120ctn), ␤-catenin, plakoglobin, APC, karyopherin ␣ (also known as importin ␣), and several other proteins (reviewed in Refs. 1-3). Nucleocytoplasmic shuttling by many ARM proteins allows them to regulate events in different cellular compartments, including gene transcription and cell adhesion (reviewed in Refs. 2-7). ARM proteins enter the nucleus by different mechanisms (reviewed in Refs. 2-7). Karyopherin ␣ enters the nucleus when it associates with proteins containing a nuclear localization signal (NLS) sequence consisting of a series of adjacent lysines or arginines (reviewed in Ref. 3). The NLS sequence is believed to anchor within the long surface groove formed by the multiple ARM repeats of karyopherin ␣, promoting the nuclear import of both the NLS-containing protein and karyopherin ␣ (3, 5). APC possesses two NLS sequences and may enter the nucleus by associating with karyopherin ␣ or related proteins (4, 7). The mechanisms by which other ARM proteins enter the nucleus are less clear, because some of these proteins neither possess classic NLS sequences, nor have they been reported to associate with NLS-containing proteins.Several ARM proteins interact with the Rho family of small GTPases (8 -15) or with guanine nucleotide exchange factors (GEFs) for these GTPases (16,17). SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), which is a series of adjacent l...

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasomemediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.

Different Shaker family alpha-subunit genes generate distinct voltage-dependent K+ currents when expressed in heterologous expression systems. Thus it generally is believed that diverse neuronal K+ current phenotypes arise, in part, from differences in Shaker family gene expression among neurons. It is difficult to evaluate the extent to which differential Shaker family gene expression contributes to endogenous K+ current diversity, because the specific Shaker family gene or genes responsible for a given K+ current are still unknown for nearly all adult neurons. In this paper we explore the role of differential Shaker family gene expression in creating transient K+ current (IA) diversity in the 14-neuron pyloric network of the spiny lobster, Panulirus interruptus. We used two-electrode voltage clamp to characterize the somatic IA in each of the six different cell types of the pyloric network. The size, voltage-dependent properties, and kinetic properties of the somatic IA vary significantly among pyloric neurons such that the somatic IA is unique in each pyloric cell type. Comparing these currents with the IAs obtained from oocytes injected with Panulirus shaker and shal cRNA (lobster Ishaker and lobster Ishal, respectively) reveals that the pyloric cell IAs more closely resemble lobster Ishal than lobster Ishaker. Using a novel, quantitative single-cell-reverse transcription-PCR method to count the number of shal transcripts in individual identified pyloric neurons, we found that the size of the somatic IA varies linearly with the number of endogenous shal transcripts. These data suggest that the shal gene contributes substantially to the peak somatic IA in all neurons of the pyloric network.

The patterned activity generated by the pyloric circuit in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, results not only from the synaptic connectivity between the 14 component neurons but also from differences in the intrinsic properties of the neurons. Presumably, differences in the complement and distribution of expressed ion channels endow these neurons with many of their distinct attributes. Each pyloric cell type possesses a unique, modulatable transient potassium current, or A-current (I A ), that is instrumental in determining the output of the network. Two genes encode A-channels in this system, shaker and shal. We examined the hypothesis that cellspecific differences in shaker and shal channel distribution contribute to diversity among pyloric neurons. We found a stereotypic distribution of channels in the cells, such that each channel type could contribute to different aspects of the firing properties of a cell. Shal is predominantly found in the somatodendritic compartment in which it influences oscillatory behavior and spike frequency. Shaker channels are exclusively localized to the membranes of the distal axonal compartments and most likely affect distal spike propagation. Neither channel is detectably inserted into the preaxonal or proximal portions of the axonal membrane. Both channel types are targeted to synaptic contacts at the neuromuscular junction. We conclude that the differential targeting of shaker and shal to different compartments is conserved among all the pyloric neurons and that the channels most likely subserve different functions in the neuron.

Val-12 after prolonged mAChR activation. We also demonstrate that Rac1 participates in mAChR-induced cell-cell compaction and c-Jun phosphorylation. These results indicate that M 3 mAChR activation converts Rac1 to the GTP-bound form, alters interactions between Rac1, IQGAP1, and actin, and causes the junctional accumulation of Rac1 and IQGAP1.The small GTPase Rac1 is emerging as an important participant in a variety of signaling pathways. Activation of Rac1 contributes to many responses in smooth muscle cells including c-Jun NH 2 -terminal kinase (JNK) 1 activation (1), reactive oxygen species generation (2), and contraction (3). The involvement of Rac1 in neuronal growth cone remodeling and neurite outgrowth implicates Rac1 as an intriguing regulator of axonal pathfinding and synaptogenesis (4 -6). The participation of Rac1 in the cadherin-mediated adhesion of epithelial and endothelial cells indicates that Rac1 signaling cascades may also regulate wound healing and vascular permeability (reviewed in Refs. 7-9). In many instances, activation of heterotrimeric G proteincoupled receptors (GPCR) initiates these Rac1-dependent cellular responses (1, 2, 5, 9). Activation of GPCR stimulates the conversion of Rac from the GDP-bound form to the GTP-bound form (1, 10). In addition to this event, GPCR activation undoubtedly induces additional uncharacterized changes in Rac1, such as altered interactions with protein partners or translocation to unique intracellular sites that promote the participation of Rac1 in these different signaling pathways. However, little is known regarding the concurrent changes in GTP binding activity, protein interactions, and subcellular localization of Rac1, which are induced by GPCR activation.The M 3 muscarinic acetylcholine receptor (mAChR) is a likely candidate to regulate Rac1 activity in a variety of cell types. The M 3 mAChR and the closely related M 1 mAChR are GPCR that are expressed in a wide variety of cells including smooth muscle cells, neurons, and epithelial and endothelial cells (reviewed in Ref. 11). Activation of M 3 mAChR induces several cellular responses that may involve Rac1 including JNK activation (12), reactive oxygen species generation (13), smooth muscle contraction (reviewed in Ref. 14), and cadherinmediated adhesion (11,15). Many of these responses have important physiological effects. For example, smooth muscle contraction induced by M 3 mAChR activation significantly alters pulmonary and cardiovascular function (reviewed in Refs. 16 and 17). The induction of E-cadherin-mediated adhesion by M 3 mAChR activation in lung carcinoma cells may diminish metastatic potential (reviewed in Refs. 11 and 15). The M 3 mAChR-mediated activation of JNK (12) may play an important role in AP-1-mediated transcription in a variety of cell types (reviewed in Ref. 18). The probability that these M 3 mAChR-dependent functions involve Rac1 provides a strong rationale for investigating how M 3 mAChR activation alters Rac1. * This work was supported by Grant R01 HL63921 from the NHLBI...

We have cloned cDNAs for the shaker potassium channel gene from the spiny lobster Panulirus interruptus. As previously found in Drosophila, there is alternative splicing at the 5Ј and 3Ј ends of the coding region. However, in Panulirus shaker, alternative splicing also occurs within the pore-forming region of the protein. Three different splice variants were found within the P region, two of which bestow unique electrophysiological characteristics to channel function. Pore I and pore II variants differ in voltage dependence for activation, kinetics of inactivation, current rectification, and drug resistance. The pore 0 variant lacks a P region exon and does not produce a functional channel. This is the first example of alternative splicing within the pore-forming region of a voltage-dependent ion channel. We used a recently identified potassium channel blocker, -conotoxin PVIIA, to study the physiological role of the two pore forms. The toxin selectively blocked one pore form, whereas the other form, heteromers between the two pore forms, and Panulirus shal were not blocked. When it was tested in the Panulirus stomatogastric ganglion, the toxin produced no effects on transient K ϩ currents or synaptic transmission between neurons.

The transient potassium current (I(A)) plays an important role in shaping the firing properties of pyloric neurons in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. The shal gene encodes I(A) in pyloric neurons. However, when we over-expressed the lobster Shal protein by shal RNA injection into the pyloric dilator (PD) neuron, the increased I(A) had somewhat different properties from the endogenous I(A). The recently cloned K-channel interacting proteins (KChIPs) can modify vertebrate Kv4 channels in cloned cell lines. When we co-expressed hKChIP1 with lobster shal in Xenopus oocytes or lobster PD neurons, they produced A-currents resembling the endogenous I(A) in PD neurons; compared with currents evoked by shal alone, their voltage for half inactivation was depolarized, their kinetics of inactivation were slowed, and their recovery from inactivation was accelerated. We also co-expressed shal in PD neurons with lobster frequenin, which encodes a protein belonging to the same EF-hand family of Ca(2+) sensing proteins as hKChIP. Frequenin also restored most of properties of the shal-evoked currents to those of the endogenous A-currents, but the time course of recovery from inactivation was not corrected. These results suggest that lobster shal proteins normally interact with proteins in the KChIP/frequenin family to produce the transient potassium current in pyloric neurons.

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