The secretin receptor is prototypic of Family B G protein-coupled receptors, a group containing several important potential drug targets. The long-term goal of this work is to better understand the structure, function, and regulation of these receptors, gaining insights that will facilitate the development of new therapeutic strategies and new drugs that can act at a variety of targets within these molecules. The proposed projects are designed to test, extend, and refine our recently-proposed molecular model of the natural agonist-occupied secretin receptor and to elucidate the molecular basis of receptor activation and regulation. There are three broad aims for this proposal.
The first aim i s designed to acquire detailed structural insights into the global structure of the intact natural agonist-occupied secretin receptor, providing insights into the molecular basis for ligand docking and propagation of conformational changes from the amino-terminal domain to the core helical bundle domain. This will be investigated using directed photoaffinity labeling, including novel dual photolabile probes, as well as using immunological probes of accessible surfaces, and testing the evolving molecular models with targeted receptor mutagenesis.
The second aim i s designed to examine the cytosolic face of the secretin receptor, focusing on molecular determinants of coupling with G proteins, the configuration of G protein coupling to the physiologic quaternary dimeric structure of this receptor, and the impact of this on receptor function. This will be investigated using receptor mutagenesis to selectively disrupt G protein coupling, confirming the structural determinants by competition with peptides from the relevant regions. Photoaffinity labeling of the protomers involved in agonist docking and the G protein coupling will be used to explore the quaternary structure of affinity states associated with receptor homo-dimers.
The third aim i s designed to explore potential strategies for the development of receptor-active drugs, including agonists, antagonists, and allosteric modulators. This will be investigated using biochemical, molecular biological and pharmacological approaches to gain insights into the detailed structural determinants for orthosteric agonists and antagonists, as well as to define the possible allosteric site of action of the endogenous agonist sequence within the receptor amino terminus. Together, these efforts should provide the finest level of molecular detail available for understanding the structure and mechanisms of ligand binding, activation, and regulation of any receptor in this receptor family.

Public Health Relevance

This project is directed to elucidate the structural basis for natural ligand binding, activation, initiation of signaling, and regulation of the secretin receptor, a prototypic member of the very important Family B group of G protein-coupled receptors. This includes biochemical, molecular biological, biophysical, and cell biological techniques. These insights will be utilized to initiate rational approaches toward the development of agonists, antagonists, and allosteric modulators acting at these receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK046577-18
Application #
8217227
Study Section
Special Emphasis Panel (ZRG1-DKUS-F (02))
Program Officer
Serrano, Jose
Project Start
1995-02-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
18
Fiscal Year
2012
Total Cost
$412,779
Indirect Cost
$154,792
Name
Mayo Clinic, Arizona
Department
Type
DUNS #
153665211
City
Scottsdale
State
AZ
Country
United States
Zip Code
85259
Miller, Laurence J; Dong, Maoqing (2013) The orthosteric agonist-binding pocket in the prototypic class B G-protein-coupled secretin receptor. Biochem Soc Trans 41:154-8
Dong, Maoqing; Pinon, Delia I; Miller, Laurence J (2013) Insights into the impact of phenolic residue incorporation at each position along secretin for receptor binding and biological activity. Regul Pept 180:5-11
Ke, Jiyuan; Harikumar, Kaleeckal G; Erice, Clara et al. (2013) Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex. Genes Dev 27:2305-19
Qi, T; Dong, M; Watkins, H A et al. (2013) Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant ýý(1-47)hCT((a)) function. Br J Pharmacol 168:644-57
Dong, Maoqing; Pinon, Delia I; Miller, Laurence J (2012) Site of action of a pentapeptide agonist at the glucagon-like peptide-1 receptor. Insight into a small molecule agonist-binding pocket. Bioorg Med Chem Lett 22:638-41
Miller, Laurence J; Dong, Maoqing; Harikumar, Kaleeckal G (2012) Ligand binding and activation of the secretin receptor, a prototypic family B G protein-coupled receptor. Br J Pharmacol 166:18-26
Ke, Jiyuan; Zhang, Chenghai; Harikumar, Kaleeckal G et al. (2012) Modulation of ?-catenin signaling by glucagon receptor activation. PLoS One 7:e33676
Garcia, Gene L; Dong, Maoqing; Miller, Laurence J (2012) Differential determinants for coupling of distinct G proteins with the class B secretin receptor. Am J Physiol Cell Physiol 302:C1202-12
Miller, Laurence J; Chen, Quan; Lam, Polo C-H et al. (2011) Refinement of glucagon-like peptide 1 docking to its intact receptor using mid-region photolabile probes and molecular modeling. J Biol Chem 286:15895-907
Koole, Cassandra; Wootten, Denise; Simms, John et al. (2011) Polymorphism and ligand dependent changes in human glucagon-like peptide-1 receptor (GLP-1R) function: allosteric rescue of loss of function mutation. Mol Pharmacol 80:486-97

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