Distinct molecular mechanisms for agonist peptide binding to types A and B cholecystokinin receptors demonstrated using fluorescence spectroscopy.
Fluorescence spectroscopy provides a direct method for evaluating the environment of a fluorescent ligand bound to its receptor. We utilized this methodology to determine the environment of Alexa within a cholecystokinin (CCK)-like probe (Alexa488-Gly-[(Nle(28,31))CCK-26-33]; CCK-8 probe) bound to the type A CCK receptor (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Here, we study this probe at the type B CCK receptor and develop another probe with its fluorophore closer to the carboxyl-terminal pharmacophore of type B receptor ligands (Alexa488-Trp-Nle-Asp-Phe-NH2; CCK-4 probe). Both probes bound to type B CCK receptors in a saturable and specific manner and represented full agonists. Similar to the type A receptor, at the type B receptor these probes exhibited shorter lifetimes and lower anisotropy when the receptor was in the active conformation than when it was shifted to its inactive, G protein-uncoupled state using guanosine 5'-[beta,gamma-imido]-triphosphate trisodium salt. Absolute values for lifetime and anisotropy were lower for the CCK-8 probe bound to the type B receptor than for this probe bound to the type A receptor, and Alexa fluorescence was more easily quenched by iodide at the type B receptor. This represents the first direct evidence that, despite having identical affinities for binding and potencies for activating type A and B receptors, CCK is docked via distinct mechanisms, with the amino terminus more exposed to the aqueous milieu when bound to the type B CCK receptor than to the type A CCK receptor. Of interest, despite this difference in binding, activation of both receptors results in analogous direction of movement of the fluorescent indicator probes.
Harikumar
Kaleeckal G
KG
Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, USA.
Clain
Jeremy
J
Pinon
Delia I
DI
Dong
Maoqing
M
Miller
Laurence J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002766:Cholecystokinin / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D007454:Iodides / Q000494:pharmacology; D008024:Ligands; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011949:Receptors, Cholecystokinin / Q000819:agonists* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007454:Iodides; D008024:Ligands; D015335:Molecular Probes; D010455:Peptides; D011949:Receptors, Cholecystokinin; D006165:Guanylyl Imidodiphosphate; D002766:Cholecystokinin