The Journal of biological chemistry
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Comparative Study
Different single receptor domains determine the distinct G protein coupling profiles of members of the vasopressin receptor family.
The vasopressin receptor family is unique among all classes of peptide receptors in that its individual members couple to different subsets of G proteins. The V1a vasopressin receptor, for example, is preferentially linked to G proteins of the Gq/11 class (biochemical response: stimulation of phosphatidylinositol hydrolysis), whereas the V2 vasopressin receptor is selectively coupled to Gs (biochemical response: stimulation of adenylyl cyclase). ⋯ On the other hand, only those hybrid receptors containing V2 receptor sequence in the third intracellular loop were capable of efficiently stimulating cAMP production. These findings suggest that the differential G protein coupling profiles of individual members of a structurally closely related receptor subfamily can be determined by different single intracellular receptor domains.
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Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. ⋯ Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.
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Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. ⋯ Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system.
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The potential for the activation of one Trk receptor by ligand binding to another Trk receptor was explored by determining if transphosphorylation on tyrosine residues can occur between receptors. For most of these experiments, functional chimeric receptors were used that contained the extracellular domain of the human type 2 tumor necrosis factor receptor and the transmembrane and cytoplasmic domains of rat TrkA, TrkB, or TrkC and that, when activated by the tumor necrosis factor, mediated the nerve growth factor-like biological activities in PC12 cells. ⋯ The failure of TrkA to take part in transphosphorylation reactions with TrkB and TrkC was confirmed using the natural receptors. Trk receptor transphosphorylation occurs in the two non-neuronal cell types, but TrkA is excluded from these reactions.
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Comparative Study
Disrupted signaling in a mutant J2E cell line that shows enhanced viability, but does not proliferate or differentiate, with erythropoietin.
The immature erythroid J2E cell line proliferates and terminally differentiates following erythropoietin stimulation. In contrast, the mutant J2E-NR clone does not respond to erythropoietin by either proliferating or differentiating. Here we show that erythropoietin can act as a viability factor for both the J2E and J2E-NR lines, indicating that erythropoietin-initiated maturation is separable from the prevention of cell death. ⋯ However, protein interactions with the erythropoietin receptor and Grb2 were restricted in the mutant cells. Subsequent investigation of several other signaling molecules exposed numerous alterations in J2E-NR cells; phosphorylation changes to phosphatidylinositol 3-kinase, phospholipase Cgamma, p120 GAP, and mitogen-activated protein kinases (p42 and p44) observed in erythropoietin-stimulated J2E cells were not seen in the J2E-NR line. These data indicate that some pathways activated during erythropoietin-induced differentiation may not be essential for the prevention of apoptosis.