Ruth Dooley

Odorant receptor heterodimerization in the olfactory system of Drosophila melanogaster

Despite increasing knowledge about dimerization of G-protein-coupled receptors, nothing is known about dimerization in the largest subfamily, odorant receptors. Using a combination of biochemical and electrophysiological approaches, we demonstrate here that odorant receptors can dimerize. DOR83b, an odorant receptor that is ubiquitously expressed in olfactory neurons from Drosophila melanogaster and highly conserved among insect species, forms heterodimeric complexes with other odorant-receptor proteins, which strongly increases their functionality.

Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1/2-dependent. Aldosterone induced the rapid activation of ERK1/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1/2 was inhibited in cells suppressed in the expression of PKD1.

The regulation of cell growth and survival by aldosterone.

The steroid hormone aldosterone is synthesized from cholesterol, mainly in the zona glomerulosa of the adrenal cortex. Aldosterone exerts its effects in the epithelial tissues of the kidney and colon and in non-epithelial tissues such as the brain and cardiovasculature. The genomic response to aldosterone involves dimerization of the mineralocorticoid receptor (MR), dissociation of heat shock proteins from MR, translocation of the aldosterone-MR complex to the nucleus and the concomitant regulation of gene expression. Rapid responses to aldosterone occur within seconds to minutes, do not involve transcription or translation and can modulate directly or indirectly the later genomic responses. Aside from the well-known effects of aldosterone on the regulation of sodium and water homeostasis, aldosterone can also produce deleterious structural changes in tissues by inducing hypertrophy and the dysregulation of proliferation and apoptosis, leading to fibrosis and tissue remodelling. Here we discuss the involvement of aldosterone-mediated rapid signalling cascades in the development of disease states such as chronic kidney disease and heart failure, and the antagonists that can inhibit these pathophysiological responses.

Aldosterone as a renal growth factor

Aldosterone regulates blood pressure through its effects on the cardiovascular system and kidney. Aldosterone can also contribute to the development of hypertension that leads to chronic pathologies such as nephropathy and renal fibrosis. Aldosterone directly modulates renal cell proliferation and differentiation as part of normal kidney development. The stimulation of rapidly activated protein kinase cascades is one facet of how aldosterone regulates renal cell growth. These cascades may also contribute to myofibroblastic transformation and cell proliferation observed in pathological conditions of the kidney. Polycystic kidney disease is a genetic disorder that is accelerated by hypertension. EGFR-dependent proliferation of the renal epithelium is a factor in cyst development and trans-activation of EGFR is a key feature in initiating aldosterone-induced signalling cascades. Delineating the components of aldosterone-induced signalling cascades may identify novel therapeutic targets for proliferative diseases of the kidney.

Protein Kinase D1 Modulates Aldosterone- Induced ENaC Activity in a Renal Cortical Collecting Duct Cell Line

Aldosterone treatment of M1-CCD cells stimulated an increase in epithelial Na(+) channel (ENaC) alpha-subunit expression that was mainly localized to the apical membrane. PKD1-suppressed cells constitutively expressed ENaCalpha at low abundance, with no increase after aldosterone treatment. In the PKD1-suppressed cells, ENaCalpha was mainly localized proximal to the basolateral surface of the epithelium both before and after aldosterone treatment. Apical membrane insertion of ENaCbeta in response to aldosterone treatment was also sensitive to PKD1 suppression as was the aldosterone-induced rise in the amiloride-sensitive, trans-epithelial current (I(TE)). The interaction of the mineralocorticoid receptor (MR) with specific elements in the promoters of aldosterone responsive genes is stabilized by ligand interaction and phosphorylation. PKD1 suppression inhibited aldosterone-induced SGK-1 expression. The nuclear localization of MR was also blocked by PKD1 suppression and MEK antagonism implicating both these kinases in MR nuclear stabilization. PKD1 thus modulates aldosterone-induced ENaC activity through the modulation of sub-cellular trafficking and the stabilization of MR nuclear localization.

The scaffold protein MUPP1 regulates odorant-mediated signaling in olfactory sensory neurons

ABSTRACT The olfactory signal transduction cascade transforms odor information into electrical signals by a cAMP-based amplification mechanism. The mechanisms underlying the very precise temporal and spatial organization of the relevant signaling components remains poorly understood. Here, we identify, using co-immunoprecipitation experiments, a macromolecular assembly of signal transduction components in mouse olfactory neurons, organized through MUPP1. Disruption of the PDZ signaling complex, through use of an inhibitory peptide, strongly impaired odor responses and changed the activation kinetics of olfactory sensory neurons. In addition, our experiments demonstrate that termination of the response is dependent on PDZ-based scaffolding. These findings provide new insights into the functional organization, and regulation, of olfactory signal transduction.

Aldosterone-induced ENaC and basal Na+/K+-ATPase trafficking via protein kinase D1-phosphatidylinositol 4-kinaseIIIβ trans Golgi signalling in M1 cortical collecting duct cells.

Aldosterone regulates Na(+) transport in the distal nephron through multiple mechanisms that include the transcriptional control of epithelial sodium channel (ENaC) and Na(+)/K(+)-ATPase subunits. Aldosterone also induces the rapid phosphorylation of Protein Kinase D1 (PKD1). PKD isoforms regulate protein trafficking, by the control of vesicle fission from the trans Golgi network (TGN) through activation of phosphatidylinositol 4-kinaseIIIβ (PI4KIIIβ). We report rapid ENaCγ translocation to the plasma membrane after 30 min aldosterone treatment in polarized M1 cortical collecting duct cells, which was significantly impaired in PKD1 shRNA-mediated knockdown cells. In PKD1-deficient cells, the ouabain-sensitive current was significantly reduced and Na(+)/K(+)-ATPase α and β subunits showed aberrant localization. PKD1 and PI4KIIIβ localize to the TGN, and aldosterone induced an interaction between PKD1 and PI4KIIIβ following aldosterone treatment. This study reveals a novel mechanism for rapid regulation of ENaC and the Na(+)/K(+)-ATPase, via directed trafficking through PKD1-PI4KIIIβ signalling at the level of the TGN.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

BackgroundExtracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation.ResultsHere we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells.ConclusionOur results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.