SUMMARY G12 class heterotrimeric G proteins stimulate RhoA activation by RGS-RhoGEFs. However, p115RhoGEF is a GTPase Activating Protein (GAP) towards Gα13 while PDZRhoGEF is not. We have characterized interaction between the PDZRhoGEF rgRGS domain (PRG-rgRGS) and the alpha subunit of G13, and determined crystal structures of their complexes in the inactive state bound to GDP, and in the active states bound to GDP·AlF (transition state) and GTPγS (Michaelis complex). PRG-rgRGS interacts extensively with the helical domain and the effector binding sites on Gα13 through contacts that are largely conserved in all three nucleotide-bound states, although PRG-rgRGS has highest affinity to the Michaelis complex. An acidic motif in the N-terminus of PRG-rgRGS occupies the GAP binding site of Gα13 and is flexible in the GDP·AlF complex, but well ordered in the GTPγS complex. Replacement of key residues in this motif with their counterparts in p115RhoGEF confers GAP activity.

Cell-cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell-cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.

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Estrogen receptor ␣ (ER␣) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ER␣ activation by estrogen up-regulates the murine Ca 2؉ -activated K ؉ channel ␣ subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ER␣ also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human smooth muscle cells. Promoter studies revealed that estrogen/hER␣-mediated hSlo1 transcription was nearly 6-fold more efficient than for mSlo1 (EC 50 , 0.07 versus 0.4 nM). Unlike the genomic transcriptional mechanism employed by mSlo1, hSlo1 exhibits a nongenomic hER␣-mediated regulatory mechanism. This is supported by the following: 1) efficient hSlo1 transcription after disruption of the DNA-binding domain of hER␣ or knockdown of Sp1, and 2) lack of AP-1 sites in the hSlo1 promoter. Three nongenomic signaling pathways were explored: Src, Rho, and PI3K. Inhibition of Src with 10 M PP2, and reported downstream ERK with 25 M PD98059 did not prevent estrogen action but caused an increase in hSlo1 basal transcription; conversely, constitutively active c-Src (Y527F) decreased hSlo1 basal transcription even preventing its estrogen/hER␣-mediated transcriptional activation. Rho inhibition by coexpressed Clostridium botulinum C3 transferase did not alter estrogen action. In contrast, inhibition of PI3K activity with 10 M LY294002 decreased estrogen-stimulated hSlo1 transcription by ϳ40%. These results indicate that the nongenomic PI3K signaling pathway plays a role in estrogen/hER␣-stimulated hSlo1 gene expression; whereas c-Src activity leads to hSlo1 gene tonic repression independently of estrogen, likely through ERK activation.Large conductance voltage-and Ca 2ϩ -activated potassium channel (MaxiK, BK) plays important roles in the regulation of vascular tone, neurotransmission, uresis, and other body functions (1-3). Disruption of its pore-forming ␣ subunit gene (Slo1) results in numerous pathologies, including ataxia, hypertension, urinary bladder incontinence, and erectile dysfunction, and has been linked to generalized epilepsy and paroxysmal dyskinesia in humans (2, 4, 5). Thus, it is relevant to investigate mechanisms that control Slo1 gene expression, especially that of the human (h) gene, hSlo1, as they may result in important therapeutic venues. In this regard, we previously showed that the murine Slo1 gene (mSlo1) expression is up-regulated by estrogen via the activation of estrogen receptor alpha (ER␣) 4 and now examine whether its human counterpart hSlo1 responds equally to estrogen.Activated estrogen receptors (ERs) can have nuclear and cytoplasmic roles; classically known as "genomic" and "nongenomic" pathways, respectively. In the nucleus, they bind directly to specific 13-bp palindromic DNA regulatory regions (with a 3-bp spacing of variable bases) of target genes known as estrogen response elements (EREs), thus...

The renin-angiotensin system (RAS) is a critical regulator of sodium balance, extracellular fluid volume, vascular resistance, and, ultimately, arterial blood pressure. The key RAS molecule angiotensin II type 1 receptor (AT1R), and another major determinant of vascular tone, the large conductance calciumactivated potassium (BK Ca ) channel, are both highly expressed in renal arterial smooth muscle cells (SMCs). Our previous studies in expression systems revealed a physical association between AT1R and BK Ca , and that AT1R association modified BK Ca channel voltage sensitivity. However, the effect of Ang II on BK Ca channels in renal arterial SMCs needs to be defined. Furthermore, whether AT1R association is critical for the alteration of channel activity in response to Ang II, and whether the coupling changes BK Ca reactivity to specific inhibitors also remain unknown. Our present studies in rat renal arterial SMCs show that application of 100 nM Iberiotoxin (IbTx, a specific BK Ca channel blocker) inhibits whole-cell BK Ca currents by 63.0512.5% (n=5). IbTxsensitive BK Ca currents were reduced by 44.458.7% (n=3) in response to 1 mM Ang II treatment. In AT1R-IRES-BK Ca -transfected HEK293T cells, extracellular application of 1 mM Ang II also suppressed whole-cell BK Ca currents by 19.750.3% (n=3), while this treatment made no significant changes in cells expressing only BK Ca (n=6). IbTx reduced the whole-cell currents by 37.251.9% (n=4) in BK Ca -transfected HEK293T cells and 37.7512.5% (n=5) in AT1R-IRES-BK Ca -transfected cells. Paxilline treatment (100 nM) produced a 66.858.0% (n=3) reduction of BK Ca whole-cell currents in BK Ca -transfected HEK293T cells and 65.859.2% (n=3) reduction in AT1R-IRES-BK Ca -transfected cells. These results demonstrate that Ang II inhibits BK Ca channel activity in renal arterial SMCs and suggest that AT1R serves as the mediator of the effect. However, the interaction between AT1R and BK Ca does not change channel reactivity towards specific channel inhibitors. Supported by NIH.