G-protein coupled receptors (GPCRs)

Class B GPCRs are peptide receptors of high pharmacological relevance to widespread diseases, such as diabetes, osteoporosis, and depression. Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating photochemical Uaa and new bioreactive Uaa probes into the intact receptor expressed in the native membrane of live cells, an unparalleled number of intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.


Figure 6.1: Photo-chemical probe Azi was incorporated into CRF1R to reveal the binding pocket (in purple) for its native peptide agonist Ucn1; Bioreactive probe Ffact was incorporated into CRF1R together with Cys in Ucn1 to determine reciprocal spatial constrains (circle with same colors) of the receptor-ligand complex. Obtained from intact receptor expressed in live cells, these data were used to build a complete conformation model for the CRF1R complexed with Ucn1, revealing its architecture and activation mechanism.

Our method represents a major advance with respect to intrinsically limited and experimentally demanding traditional photo-crosslinking approaches, and provides unique panoramic information derived from the full-length receptor in the native context of the live cell, which complements data derived from crystallographic characterization of isolated receptors in an artificial environment. This strategy should be generally applicable to study protein interactions under native conditions.