A SEGMENT OF 5 AMINO-ACIDS IN THE 2ND EXTRACELLULAR LOOP OF THE CHOLECYSTOKININ-B RECEPTOR IS ESSENTIAL FOR SELECTIVITY OF THE PEPTIDE AGONIST GASTRIN

The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin, CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein coupled receptors having approximately 50% amino acid homol ogy. Both the CCKAR and CCKBR have high affinity for sulfated CCK pept ides, while only the CCKBR has high affinity for gastrin peptides. To determine the structural basis for the selectivity of the CCKBR for ga strin, we first constructed a series of CCKB/AR chimeras in which rest riction endonuclease-defined segments of the CCKBR were replaced with the corresponding segments of the CCKAR. Chimeras transiently expresse d in COS-1 cells were screened for the selective loss of gastrin affin ity according to the displacement of I-125-labeled Bolton-Hunter-CCK-8 binding by gastrin-17-I and CCK-8. The sequence spanning from transme mbrane domain III (TM III) to TM. V was the only segment that resulted in the selective loss of gastrin affinity. This segment could account for 100 of the expected 300-fold lower affinity of gastrin-17-I obser ved for the control CCKAR compared to the control CCKBR. Using site-di rected mutagenesis in this segment of the CCKBR, we identified a seque nce of 5 amino acids in the second extracellular loop responsible for this 100 fold selective loss in gastrin affinity, I-125-labeled Bolton -Hunter-CCK-8 binding displacement by L365,260 (a CCKBR selective anta gonist) was unaffected by the changes in these 5 amino acids. These re sults present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for g astrin.