Ligand-Receptor Interactions Regulating Cell Trafficking and Signaling of the Human PTH1Rc: An in Silica Examination

Desensitization of G-protein coupled receptors is a fundamental mechanism regulating the cellular responsiveness to agonists. It was recently demonstrated that stimulation of the human parathyroid hormone (PTH)/ parathyroid hormone related protein (PTHrP) receptor (PTH1Rc) by its cognate agonists triggers the rapid beta-arrestin2-dependent internalization of ligand--receptor complexes. In turn, the beta-arrestin2--receptor interaction results in rapid desensitization of intracellular signaling as measured by reduced cAMP levels. Recently, we reported that Bpa1-PTHrP(1-36), a Gs-signaling-selective agonist, does not stimulate mobilization of b-arrestin2 nor PTH1Rc internalization. Bpa1-PTHrP(1-36)-mediated recruitment of b-arrestin2 is restored by a single Ala point-mutation in transmembrane helix 6. Molecular models were developed for wild type and Ala mutant PTH1Rc--ligand complexes, with the aim of characterizing the different modes of ligand binding that may explain the above. Conformational features of numerous receptor fragments, determined by CD and NMR, were incorporated in the models, as well as the arrangement of transmembrane helices based on the X-ray structure of rhodopsin and the contact points identified by photoaffinity crosslinking studies. These models were utilized as starting structures for MD simulations. The analysis of the ligand-induced transmembrane domains motions indicates that substitution of Bpa for Ser1 in the ligand leads to major changes in the relative positions of transmembrane helices. In turn, this relative displacement in the transmembrane domains results in a conformational change in the 3rd cytosolic loop, known to be involved in the interaction with the G protein. These changes can be traced back to different sets of interactions between specific residues of the ligand and of the receptor. In conclusion, the molecular models described here allow for an atomic insight into the structure of agonist-bound PTH1Rc interacting with beta-arrestin2.