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Activation of MAPK by TRH Requires Clathrin-Dependent Endocytosis and PKC but Not Receptor Interaction with ß-Arrestin or Receptor Endocytosis

Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Address all correspondence and requests for reprints to: Dr. Patricia M. Hinkle, Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. E-mail: Patricia_Hinkle{at}urmc.rochester.edu

To determine whether the interaction of the TRH receptor with ß-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a ß-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the ß-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the ß-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the ß-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the ß-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind ß-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind ß-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.

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