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J Philie

Publications and source records attributed to J Philie.

6 recordsLinked to original sources
PubMed1999

The N terminus of amphiphysin II mediates dimerization and plasma membrane targeting.

Amphiphysin I and II are nerve terminal-enriched proteins containing SH3 domains that interact with dynamin and synaptojanin. The amphiphysins may function in synaptic vesicle endocytosis by targeting synaptojanin and dynamin to emerging endocytic buds through SH3 domain-independent interactions with clathrin and AP2. We have recently identified and cloned several amphiphysin II splice variants that differentially incorporate clathrin-binding domains. To determine whether these domains function in membrane targeting, we used immunofluorescence to examine the potential localization of amphiphysin II variants to clathrin-coated pits on plasma membranes purified from transfected COS-7 cells. Full-length amphiphysin II targets to the plasma membrane where it partially co-localizes with clathrin. However, splice variants and deletion constructs lacking clathrin-binding domains still target to the plasma membrane, and removal of clathrin from the membrane does not affect amphiphysin II distribution. Surprisingly, plasma membrane targeting was dependent on the presence of a 31-amino acid alternatively spliced sequence at the N terminus of amphiphysin II, a result confirmed using subcellular fractionation. In binding assays, the 31-amino acid sequence was also found to facilitate amphiphysin dimerization mediated through the N terminus. Taken together, these data support a role for the N terminus of amphiphysin II in membrane targeting during endocytosis.

Alternative Splicing↗
PubMed1997

Modulation of alpha7 nicotinic receptor-mediated calcium influx by nicotinic agonists.

Our previous work had demonstrated stable expression of rat alpha7 alpha-bungarotoxin (alpha-BGT) binding sites by GH4C1 rat pituitary cells, a clonal line that does not endogenously express nicotinic receptors. The stably expressed alpha7 sites had similar binding affinities, pharmacological profiles, kinetic properties, and molecular size as rat brain alpha-BGT receptors, suggesting that they represent a good model system for studying receptor function. The present data show that nicotinic receptor agonists increase intracellular calcium levels ([Ca2+]i), as assessed using Fura-2, in alpha7/GH4C1 cells in a dose-dependent manner with EC50 values that correlate well with the affinity of these ligands for alpha7/GH4C, alpha-BGT receptors. Nicotinic receptor antagonists inhibited agonist-induced increases in [Ca2+]i, with IC50 values in the nanomolar to micromolar range. The nicotinic agonist-induced increase in [Ca2+]i required extracellular calcium and did not occur in the presence of CdCl2, suggesting that agonist-induced increases in [Ca2+]i are due to an influx of extracellular calcium through voltage-gated calcium channels. Preexposure of the alpha7/GH4C1 cells to 8-bromo cAMP resulted in an enhanced [Ca2+]i in response to agonist, suggesting that phosphorylation by adenylate cyclase may regulate receptor responsiveness. Interestingly, short-term preexposure (40-60 sec) of the cells to subthreshold concentrations of nicotinic agonist-enhanced receptor-stimulated calcium influx (up to 55%) while activating agonist concentrations completely blocked receptor-mediated responses. Long-term exposure of alpha7/GH4C1 cells to K+ resulted in about a 2-fold increase in alpha-BGT receptors and in agonist-evoked calcium influx. The sensitivity of these up-regulated receptors were modulated by subthreshold and activating concentrations of agonist in a manner similar to control receptors. The present results, demonstrating a biphasic regulation of alpha7 receptor-mediated calcium influx by nicotinic agonists, suggest that these receptors may play an important role in neuronal function under control and depolarizing conditions.

Animals↗
PubMed1993

Functional nicotinic receptor expression in mesodermal cells transfected with MyoD cDNA.

Previous studies had shown that MyoD promoted nicotinic acetylcholine subunit gene expression; the present experiments were done to determine whether this subsequently led to the development of functional nicotinic acetylcholine receptors. Transfection of C3H 10T1/2 cells with MyoD cDNA resulted in the appearance of [125I]alpha-bungarotoxin binding sites; radiolabelled alpha-toxin binding was not observed in cells transfected with a plasmid that lacked MyoD cDNA. Receptor development plateaued over a time course of several days with maximal binding seven and 11 days after exposure to fusion medium. [125I]alpha-bungarotoxin binding was of high affinity (Kd = 1 nM), saturable and was inhibited by nicotinic but not muscarinic receptor ligands, with IC50s of 1-3 nM for alpha-bungarotoxin, 1-3 microM for d-tubocurarine and 3-10 microM for nicotine. Not only did the cells exhibit a cell surface nicotinic receptor but they also expressed a nicotinic receptor mediated functional response. Carbachol resulted in uptake of 22Na into the cells at concentrations similar to those required for receptor activation at a muscle type nicotinic receptor; furthermore, the functional response was effectively blocked by nicotinic receptor ligands, including alpha-bungarotoxin (IC50 = 2 to 6 nM) and d-tubocurarine (IC50 = 0.1 to 0.4 microM); muscarinic receptor ligands had no effect. A time course study showed that alpha-bungarotoxin binding and carbachol stimulated 22Na uptake developed in parallel, suggesting that the observed functional response was mediated through an interaction at the alpha-bungarotoxin recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Thymopoietin, a thymic polypeptide, prevents nicotinic agonist-induced morphological changes in neonatal muscle cells in culture.

Thymopoietin, a polypeptide hormone isolated from thymus and involved in immune function, potently inhibited [125I]alpha-bungarotoxin binding to neonatal muscle cells in culture (IC50 = 3.8 nM) and blocked carbachol-stimulated 22Na uptake with an IC50 of 1.9 +/- 0.2 nM and 23 +/- 7 nM at a half-maximal and maximal concentration of carbachol, respectively. Studies were subsequently done to evaluate potential long-term functional consequences of this interaction of thymopoietin at the nicotinic receptor. Exposure (1-3 days) of neonatal muscle cells in culture to nicotine (3 x 10(-6) M) or carbachol (1 x 10(-6) M) resulted in a decline in myotube branching and a decrease in myotube length. Thymopoietin did not appreciably alter myotube morphology on its own; however, it prevented the effects of nicotine and carbachol on muscle cell morphology at concentrations (1-10 nM) which corresponded well to those with which thymopoietin interacted at the receptor. The action of alpha-bungarotoxin on the myotubes was very similar to that of thymopoietin. These studies suggest that the endogenously occurring polypeptide, thymopoietin, has the potential to modulate muscle cell morphology through an interaction at the nicotinic receptor.

Analysis of Variance↗
PubMed1987

Dopamine D2 receptor binding in adrenal medulla: characterization using [3H]spiperone.

The possibility that dopamine may function as a neuromodulator or neurotransmitter in the adrenal gland, and not merely serve as a precursor to the catecholamines, has been suggested. If this hypothesis is correct, receptors for dopamine should be identifiable in the adrenal. The present work demonstrates the existence of a high-affinity receptor in adrenal medulla using [3H]spiperone as the radioligand to label the receptors. [3H]Spiperone bound rapidly, reversibly, and with high affinity to bovine adrenal medullary membranes. Scatchard analysis yielded a Kd of 0.09 nM and a Bmax of 51 fmol/mg protein. In competition binding experiments, dopaminergic antagonists were at least 100 times more potent in displacing [3H]spiperone from its binding sites than adrenergic or serotonergic receptor antagonists. Similarly, agonists at the dopamine receptor more readily competed for [3H]spiperone binding than other receptor agonist drugs tested. Furthermore, D2 selective antagonists and agonists were much more potent than D1 receptor ligands. These results suggest that [3H]spiperone may bind to a high-affinity D2 dopamine receptor in adrenal medulla.

Adrenal Medulla↗