Carboxyl-terminal domains determine internalization and recycling characteristics of bombesin receptor chimeras

To investigate the role of the carboxyl terminus in the regulation of the bombesin (BN) receptor, we constructed two chimeric receptors with carboxyl termini transferred from either m3 muscarinic cholinergic (m3 ACh) (BMC) or cholecystokinin A (CCK(A)) (BCC) receptors and expressed them in Chinese hamster ovary cells. Previous studies showed that agonist treatment caused rapid internalization of CCK(A) but not m3 ACh receptors in these cells. In the current study we conducted separate analyses of ligand and receptor internalization and analyzed receptor recycling. Ligand internalization was assessed using acid washing. BN and CCK(A) receptors internalized ligand with 80 ± 3 and 85 ± 7% in an acid-resistant compartment at equilibrium. Ligand internalization of chimeric receptors generally assumed the properties of the donor receptors. Thus, BCC receptors internalized ligand to a similar extent as wild-type CCK(A) receptors (75 ± 3%), whereas, BMC receptors showed reduced ligand internalization (38 ± 1%). Receptor internalization was more directly assessed by determining agonist-induced loss of surface binding. BN and CCK(A) receptors were largely internalized (56 ± 8 and 50 ± 7%, respectively). BCC receptors were also extensively internalized (82 ± 3%). In contrast, BMC receptors were minimally internalized (22 ± 8%). Receptor recycling was assessed as recovery from agonist induced loss of binding. BN, CCK(A), and BMC receptors showed rapid recycling. In contrast, BCC receptors did not recycle. These data indicate that carboxyl-terminal structures determine both internalization of ligand-receptor complexes and subsequent receptor recycling.