8 research outputs found
Conformational Changes in the Parathyroid Hormone Receptor Associated with Activation by Agonist
Binding of hormones to their cognate G protein-coupled receptors (GPCRs) induces conformational shifts within the receptor based on evidence from a few hormone-receptor systems. Employing an engineered disulfide bond formation strategy and guided by a previously established model of the PTH-PTH receptor (PTHR)1 bimolecular complex, we set out to document and characterize the nature of agonist-induced changes in this family B GPCR. A mutant PTHR1 was generated which incorporates a Factor Xa cleavage site in the third intracellular loop. Treatment with Factor Xa fragments the receptor. However, if a new disulfide bond was formed before exposure to the enzyme, the fragments remain held together. A set of double cysteine-containing mutants were designed to probe the internal relative movements of transmembrane (TM) helices 2 and TM7. PTH enhanced formation of disulfide bonds in the K240C/F447C and A242C/F447C mutants. For the F238C/F447C mutant, a disulfide bond is formed in the basal state, but is disrupted by interaction with PTH. For the D241C/F447C PTHR1 construct, no disulfide bond formation was observed in either the basal or hormone-bound state. These findings demonstrate that the conformation of PTHR1 is altered from the basal state when PTH is bound. Novel information regarding spatial proximities between TM2 and TM7 of PTHR1 and the nature of relative movements between the two transmembrane regions was revealed. The data confirm and extend the experimentally derived model of the PTH-PTHR1 complex and provide insights at a new level of detail into the early events in PTHR1 activation by PTH
Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor
Recommended from our members
A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity
The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely even in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided dual potency/selectivity structure-activity relationships from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 (DPP4) inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling, and suggest such profiling can be incorporated into the earliest stages of drug discovery
Recurrent and novel disease-causing F8 variants in boys with severe haemophilia A in Poland
A General Method for Making Peptide Therapeutics Resistant to Serine Protease Degradation: Application to Dipeptidyl Peptidase IV Substrates
Bioactive peptides have evolved to
optimally fulfill specific biological
functions, a fact which has long attracted attention for their use
as therapeutic agents. While there have been some recent commercial
successes fostered in part by advances in large-scale peptide synthesis,
development of peptides as therapeutic agents has been significantly
impeded by their inherent susceptibility to protease degradation in
the bloodstream. Here we report that incorporation of specially designed
amino acid analogues at the P1′ position, directly C-terminal
of the enzyme cleavage site, renders peptides, including glucagon-like
peptide-1 (7–36) amide (GLP-1) and six other examples, highly
resistant to serine protease degradation without significant alteration
of their biological activity. We demonstrate the applicability of
the method to a variety of proteases, including dipeptidyl peptidase
IV (DPP IV), dipeptidyl peptidase 8 (DPP8), fibroblast activation
protein α (FAPα), α-lytic protease (αLP),
trypsin, and chymotrypsin. In summary, the “P1′ modification”
represents a simple, general, and highly adaptable method of generating
enzymatically stable peptide-based therapeutics