Abstract
The receptor for the protease thrombin is a member of the G protein-coupled receptor family, but is activated by a unique proteolytic mechanism. The irreversibility of this proteolytic mechanism and the fact that the ligand is tethered to its receptor raise special questions about inactivation of cleaved receptors and recovery of thrombin responsiveness. We compared the intracellular trafficking of the thrombin receptor to that of the beta 2-adrenergic receptor in transfected Rat1 fibroblasts. In unstimulated cells almost all beta 2 receptors were located on the plasma membrane; by contrast, part of a cell's thrombin receptors were found in an intracellular membrane compartment which co-localized with Golgi markers. Stimulation by agonist caused internalization and subsequent recycling of the beta 2-adrenergic receptor, but most activated thrombin receptors were internalized and targeted to lysosomes. The intracellular pool of thrombin receptors found in unstimulated cells was protected from activation by thrombin, but was translocated to the plasma membrane upon activation of cell surface thrombin receptors. Replenishment of plasma membrane thrombin receptors correlated with recovery of thrombin responsiveness. These observations reveal a novel trafficking mechanism for resensitizing the thrombin receptor as opposed to the internalization/recycling pathway of other G protein-coupled receptors.
Highlights
From the $Division of Cardiovascular Medicine, $$Howard Hughes Medical Institute, Stanford University Medical School, Stanford
The irreversibility membrane microdomains may alsocontribute to thespecificity of this proteolytic mechanism and the fact that the li- of receptor, G protein, and effector interactions(Neerand gand is tethered to its receptor raise special questions Clapham, 1988)
Stimulation by agonist caused internalizationto effect receptor activation. This irreversible proteolytic actiand subsequent recyclingof the &-adrenergic receptor, vation mechanism stands in contrast to thereversible ligandbut most activated thrombin receptors were internal- ing mechanisms utilized by other members of the G proteinized and targetedto lysosomes.The intracellular poolof thrombin receptors founindunstimulatedcellswas protected from activation bythrombin,butwastranslocoupled receptor family and suggests profound differences in desensitization and resensitization pathwayfsor thrombin recated to the plasma membrane upon activation of cell ceptor signaling
Summary
From the $Division of Cardiovascular Medicine, $$Howard Hughes Medical Institute, Stanford University Medical School, Stanford. Receptor Phosphorylation-Phosphorylation experiments were peronstrated previously (Ishiiet al., 19931, briefexposure t o formed as described before (Ishii et al, 1994)B. riefly, transfected R a t l thrombin resulted in a complete loss of cell surface binding of fibroblasts on six-well plates were labeledfor 60 min a t 37 "C with 0.2 mCi of [32Plorthophosphate.
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