Real-time visualization of processive myosin 5a-mediated vesicle movement in living astrocytes.

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

Rapid desensitization of the TRH receptor and persistent desensitization of its constitutively active mutant.

We studied rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R) or the m1-muscarinic receptor (m1-R) to a short challenge of threshold TRH concentration and persistent desensitization due to constitutive activity of a mutant TRH-R. Xenopus oocytes expressing TRH-Rs and/or m1-Rs were challenged for 15 s with threshold concentrations of TRH ([TRH]) and then immediately with supraoptimal [TRH] or acetylcholine ([ACh]). The threshold challenge caused desensitization of 50 - 57% of responses to subsequent supraoptimal stimulation with TRH or ACh. The homologous desensitization was reversible within 60 s after removal of the agonist. The protein kinase C (PKC) inhibitor, chelerythrine, inhibited the control responses by 30 - 40%, without affecting the desensitized responses. Chelerythrine or the phosphatase inhibitor, okadaic acid, had little effect on the kinetics of resensitization, indicating limited involvement of PKC. In oocytes coexpressing wild type TRH-Rs or m1-Rs with a constitutively active TRH-R mutant (C335Stop TRH-R), a persistent desensitization (33 - 57%) of the responses to TRH or ACh was observed. Additionally, there was a complete loss of the rapid desensitization induced by threshold [TRH]. Chlorodiazepoxide (CDE), a competitive binding antagonist of TRH-Rs and an inverse agonist of C335Stop TRH-Rs, abolished the persistent desensitization induced by C335Stop TRH-Rs and enabled the rapid desensitization, conferring the wild type phenotype on C335Stop TRH-Rs. Chelerythrine had qualitatively the same effect as CDE. In conclusion, unlike the rapid desensitization, the persistent desensitization caused by the constitutively active C335Stop TRH-Rs is largely mediated by PKC. It abrogates, however, the rapid desensitization, suggesting a common mechanistic step(s).