LIGAND-STIMULATED BETA(2)-ADRENERGIC RECEPTOR INTERNALIZATION VIA THECONSTITUTIVE ENDOCYTIC PATHWAY INTO RAB5-CONTAINING ENDOSOMES

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytos is. However, it is unknown whether rab5 regulates receptors whose inte rnalization is stimulated by the binding of ligand, and whether such r eceptors change the underlying rate of the endocytic pathways they uti lize, As a model for ligand-stimulated endocytosis, we used transfecte d HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor, Nearly all receptors were on the cell surface i n the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5, H ypertonic sucrose blocked beta(2)-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediat ed process, In contrast, an inhibitor of potocytosis had little effect upon beta(2)-adrenergic receptor internalization, suggesting that thi s process did not require active caveolae, Consistent with this findin g, caveolin was not detectable in the 12 beta 6 line, as assessed by w estern blotting with a polyclonal anti-caveolin antibody, Stimulated r eceptor internalization did not affect the rate or capacity of the con stitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor, Al together, these data suggest that beta(2)-adrenergic receptors interna lize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway, Further, agonist-stimulated receptor internalization has no d etectable effect upon the function of this pathway.