Agonist stimulation of the beta-adrenergic receptor (BAR)-coupled adenylyl cyclase leads not only to activation and formation of cyclic AMP, but also to adaptation and attenuation of the response. At least three major mechanisms of receptor regulation have been identified: desensitization, internalization, and down- regulation. We and others have found that the three human beta-subtypes, B1AR, B2AR and B3AR, differ in their ability to be regulated. B2AR is the most susceptible, B1AR is less susceptible and B3AR is resistant to regulation. We have been comparing the internalization of B1AR and B2AR. Previously we showed that when each subtype is stably expressed in the same cell line, B1AR undergoes less agonist-mediated internalization than B2AR. Both G protein-coupled receptor kinases (GRKs) and arrestins (ARRs) have been implicated in B2AR internalization. The GRK-phosphorylated B2AR binds ARR which acts as an adapter between the receptor and clathrin. Endocytosis of B2AR also is dependent on dynamin, a GTPase involved in pinching off of clathrin-coated vesicles. We found that overexpressing either GRK or ARR increased the rate and extent of B1AR internalization, ARR being more effective than GRK, and B1AR being more affected than B2AR. Thus, cells overexpressing ARR internalized both subtypes at similar rates and extents. Furthermore, when we co-expressed BAR and dominant-negative mutants of dynamin or ARR, we observed that internalization of both subtypes was blocked. To visualize the endocytosis pathway used by B1AR, we recently constructed a green fluorescent protein (GFP)-tagged B1AR and stably expressed it in baby hamster kidney cells. After establishing that B1AR-GFP had properties similar to wild-type B1AR, we used confocal fluorescent microscopy to follow the agonist-mediated internalization of B1AR-GFP in viable cells. In unstimulated cells, B1AR-GFP was predominantly localized to the cell surface; but upon agonist exposure, some disappeared from the cell surface and appeared in cytoplasmic structures characteristic of endosomes. In cells loaded with fluorescent-labeled transferrin, an endosomal marker, we observed colocalization of both transferrin and B1AR-GFP in some of the same endosomes. Both the redistribution of B1AR-GFP and its colocalization with transferrin were increased in cells cotransfected with ARR. Even though B1AR undergoes less agonist-mediated internalization than B2AR, it follows the same clathrin-mediated endocytic pathway as B2AR. We also discovered that following agonist-mediated internalization of B1AR, the receptor did not recycle back to the cell surface upon removal of the agonist. This is in marked contrast to B2AR, which does recycle in cells washed free of agonist. B1AR recycling, however, was observed in cells overexpressing either GRK or ARR. These results indicate that the internalization and trafficking of B1AR differ from that of B2AR. We believe that the difference is a result of B1AR being a poorer target for GRKs and ARRs compared to B2AR.
