Structure-activity function for binding and signaling in CHO-K1 and COS-7 cells expressing the cholecystokinin A receptor.
Key amino acids of the cholecystokinin (CCK) peptide for receptor binding are sulfated Y27, W30, D32, and F33-NH(2). Three-dimensional modeling showed that the CCK-A receptor (CCK-AR) antagonist devazepide penetrated into the transmembrane (TM) domains, whereas CCK was placed on the surface of the CCK-AR. Four types of rat CCK-AR cDNAs were transfected into CHO-K1 and COS-7 cells: normal CCK-AR cDNA transfected cells (wild type, WT); K120 substituted with V; K130V; and R352V. Binding of [3H]CCK-8 was observed in WT and K130V, but not in K120V and R352V. CCK caused Ca(2+) spiking in WT and K130V, whereas K120V and R352V had no effect. Three chimeras including the CCK-AR/3ibeta2 adrenergic receptor (beta2AR), 3Nibeta2AR, and 3Cibeta2AR were constructed. Two groups of point mutations in the CCK-AR3i were also made: Y252V, S274V, S281V, and S289V (non-phospho-acceptor Y or S); S260V, S264V, S271V, and S275V (phospho-acceptor S). WT and CCK-AR/3Cibeta2AR increased [Ca(2+)](i) in response to CCK; 3Nibeta2AR was vice versa. CCK failed to increase [IP(3)] in phospho-acceptor S to V without affecting binding. Non-phospho-acceptor S or Y to V showed normal response. Thus, Lys120 outside the TM2 and Arg352 outside the TM6 of the CCK-AR are amino acids interacting with Tyr[SO(3)H]27 and Asp32 of the CCK peptide for binding. Phospho-acceptor Ser groups in the CCK-AR 3Ni are amino acids for initiating cell signaling.
Tsunoda
Yasuhiro
Y
Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA. ytsunoda@umich.edu
Song
Il
I
Taylor
Larry P
LP
Owyang
Chung
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D020109:Devazepide / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011869:Radioligand Assay; D051381:Rats; D043886:Receptor, Cholecystokinin A / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D012844:Sincalide / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014162:Transfection
D010446:Peptide Fragments; D012333:RNA, Messenger; D043886:Receptor, Cholecystokinin A; C025997:cholecystokinin (27-33); D015544:Inositol 1,4,5-Trisphosphate; D020109:Devazepide; D012844:Sincalide; D002118:Calcium