Xi Qin Ding

Disulfide bond structure and accessibility of cysteines in the ectodomain of the cholecystokinin receptor : Specific mono-reactive receptor constructs examine charge-sensitivity of loop regions

Cysteine residues play a unique role in structural analysis. We examined endogenous cysteine residues in the cholecystokinin receptor to determine participation in disulfide bonds and accessibility to methanethiosulfonate (MTS) reagents. Bonds linking Cys114 to Cys196 and Cys18 to Cys29 were demonstrated, with the first functionally important and the amino-terminal bond having no apparent function. Cys94, in the second transmembrane segment, was also accessible. Mutation of this residue to serine (C94S) was key for establishing a null cysteine-reactive pseudo-wild type receptor that could act as a template for insertion of a reactive cysteine (N102C, A204C, and T341C). Modification of T341C with a negatively charged MTS reagent reduced CCK agonist binding, while this binding was enhanced by a positively charged MTS reagent. This pattern was repeated in mutants having the same residue directly replaced with a charged residue.

Characterization of the type A cholecystokinin receptor hormone-binding domain: use of contrasting and complementary methodologies

Insights into the molecular basis of binding of the peptide hormone, cholecystokinin, to its G protein-coupled receptor is of substantial interest and may contribute to the successful production and refinement of receptor-active drugs. A number of methodological approaches provide complementary data to contribute to these insights. These include receptor mutagenesis, ligand structure-activity data, conformational analysis of ligand and receptor fragments, and photoaffinity labeling. In this work, we compare and contrast each of these methods and provide our current view of the cumulative impact of the current data on molecular conformational models of the agonist-occupied type A cholecystokinin receptor. These support the key roles played by extracellular loop and tail regions of this receptor for binding its natural peptide ligand.

Insights into the molecular basis of ligand binding by the cholecystokinin receptor

The receptor for the peptide hormone, cholecystokinin, is a G-protein-coupled receptor in the rhodopsin/β-adrenergic receptor family. A number of methodological approaches have been utilized to gain insights into the molecular basis for natural peptide ligand binding and activation of this physiologically important receptor. Insights into this have come from sequence analysis, ligand and receptor structure-activity data, receptor mutagenesis, conformational analysis of ligand and receptor fragments, and photoaffinity labeling. In this work, we review the contributions of each of these complementary approaches and provide a current integrated view of the active complex of cholecystokinin bound to its receptor.

Receptor biology and signal transduction

This year has witnessed substantial advances in receptor biology and signal transduction that are relevant to the function and regulation of the healthy pancreas and to the pathogenesis and potential therapy of pancreatitis and pancreatic carcinoma. There has been an expansion in the cast of pancreatic regulatory molecules, now including protease-activated receptors, chemokines, and chemokine receptors. There have been new insights into the cellular distribution and signaling initiated at the classic pancreatic receptors. There have also been dramatic advances in insights into the structure of G protein-coupled receptors, with the first solution of a crystal structure of a member of this superfamily, and into the molecular basis of ligand binding and activation of these important molecules. This will clearly improve the opportunities for the rational design and refinement of receptor-active drugs. In addition to these fundamental advances, there has been renewed attention to the expression, function, and regulation of receptors and signaling pathways in pancreatic cells present in the setting of pancreatitis and pancreatic carcinoma. It is hoped that this will contribute toward earlier diagnosis, more successful therapy, and new chemopreventive strategies for these illnesses.