G protein-coupled
receptors (GPCRs) represent the largest class
of receptors involved in signal transduction across cell membranes
and are major drug targets in all clinical areas. Endocytosis of GPCRs
offers a regulatory mechanism for sustaining their signaling within
a stringent spatiotemporal regime. In this work, we explored agonist-induced
endocytosis of the human serotonin1A receptor stably expressed
in HEK-293 cells and the cellular machinery involved in receptor internalization
and intracellular trafficking. The serotonin1A receptor
is a popular GPCR implicated in neuropsychiatric disorders such as
anxiety and depression and serves as an important drug target. In
spite of its pharmacological relevance, its mechanism of endocytosis
and intracellular trafficking is less understood. In this context,
we have utilized a combination of robust population-based flow cytometric
analysis and confocal microscopic imaging to address the path and
fate of the serotonin1A receptor during endocytosis. Our
results, utilizing inhibitors of specific endocytosis pathways and
intracellular markers, show that the serotonin1A receptor
undergoes endocytosis predominantly via the clathrin-mediated pathway
and subsequently recycles to the plasma membrane via recycling endosomes.
These results would enhance our understanding of molecular mechanisms
of GPCR endocytosis and could offer novel insight into the underlying
mechanism of antidepressants that act via the serotonergic pathway.
In addition, our results could be relevant in understanding cell (or
tissue)-specific GPCR endocytosis.
Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.
A series of "long-chain" 1-(2-methoxyphenyl)piperazine derivatives containing an environment-sensitive fluorescent moiety (4-amino-1,8-naphthalimide, 4-dimethylaminophthalimide, dansyl) was synthesized. The compounds displayed very high to moderate 5-HT(1A) receptor affinity and good fluorescence properties. 6-Amino-2-[5-[4-(2-methoxyphenyl)-1-piperazinyl]pentyl]-1H-benz[de]isoquinoline-1,3(2H)-dione (4) combined very high 5-HT(1A) receptor affinity (K(i) = 0.67 nM), high fluorescence emission in CHCl(3), and undetectable fluorescence emission in aqueous solution. It was evaluated for its ability to visualize 5-HT(1A) receptors overexpressed in CHO cells by fluorescence microscopy.
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