Biomedical subjects

Andra S Stevenson

Publications and source records attributed to Andra S Stevenson.

4 recordsLinked to original sources

PubMed2004

The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle.

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.

Blotting, Western↗

PubMed2003

Constitutively elevated nuclear export activity opposes Ca2+-dependent NFATc3 nuclear accumulation in vascular smooth muscle: role of JNK2 and Crm-1.

The transcription factor NFAT (nuclear factor of activated T-cells) is a cytosolic phosphoprotein that accumulates in the nucleus following dephosphorylation by the calcium (Ca2+)/calmodulin-dependent phosphatase, calcineurin. A defining feature of stimuli that induce NFAT nuclear accumulation/activation is a sustained increase in global intracellular Ca2+. Contrary to expectations, we have found that a sustained elevation of intracellular Ca2+, induced by membrane potential depolarization and mediated by voltage-dependent Ca2+ channels, does not result in nuclear localization of the NFATc3 isoform in smooth muscle. However, vasoconstrictors (e.g. uridine triphosphate (UTP)) and growth factors, which elevate intracellular Ca2+ and engage multiple intracellular signaling pathways, induce a robust increase in smooth muscle nuclear NFATc3. Here we show that depolarizing stimuli that normally fail to induce NFATc3 nuclear accumulation in arterial smooth muscle effectively induce nuclear accumulation under conditions in which Crm-1-dependent or JNK2-mediated nuclear export processes are disrupted. Consistent with an important regulatory role for JNK, UTP exerts a suppressive effect on JNK activity in smooth muscle. Export of nuclear NFATc3 following UTP-induced nuclear accumulation is dramatically slowed in cerebral arteries from JNK2-/- animals. These data indicate that in smooth muscle, stimulation of Ca2+-dependent, calcineurin-mediated nuclear import and suppression of Crm-1/JNK-dependent nuclear export are both required for induction of NFATc3 nuclear accumulation. These results highlight the dynamic interplay between influences that promote and oppose NFAT nuclear accumulation and suggest that in arterial smooth muscle suppression of constitutive nuclear export activity is an important property of NFAT-activating stimuli.

Active Transport, Cell Nucleus↗

PubMed2003

NFAT regulation in smooth muscle.

First identified in activated T cells, the calcium (Ca2+)-dependent transcription factor, nuclear factor of activated T cells (NFAT), has since been shown to play a role in nonimmune cells, including cells of the cardiovascular system. In arterial smooth muscle, the diverse array of calcium-signaling modalities, the functional interplay between smooth muscle and endothelial cells, and the influence of intravascular pressure on calcium and other signaling pathways creates a calcium-regulatory environment that is arguably unique. This review focuses on mechanisms that control the initial Ca2+/calcineurin-dependent events in NFAT activation, with a particular emphasis on NFAT regulation in native vascular smooth muscle. Also addressed is the role of additional mechanisms that act to modulate calcineurin-dependent NFAT nuclear import/export, mechanisms that may have particular relevance in this tissue.

Cardiomegaly↗

PubMed2002

Opposing actions of inositol 1,4,5-trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle.

The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca(2+)-dependent gene transcription in diverse cell types outside of the immune system. We have previously shown that nuclear accumulation of NFATc3 is induced in ileal smooth muscle by platelet-derived growth factor in a manner that depends on Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Here we show that NFATc3 is also the predominant NFAT isoform expressed in cerebral artery smooth muscle and is induced to accumulate in the nucleus by UTP and other G(q/11)-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca(2+) release through inositol 1,4,5-trisphosphate receptors and extracellular Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca(2+) influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca(2+) release by ryanodine receptors in the form of Ca(2+) sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca(2+) sparks. These results are consistent with a multifactorial regulation of NFAT nuclear accumulation in smooth muscle that is likely to involve several intracellular signaling pathways, including local effects of sarcoplasmic reticulum Ca(2+) release and effects attributable to global elevations in intracellular Ca(2+).

Active Transport, Cell Nucleus↗