Abstract
We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.
Highlights
NTSR1 to relate agonist binding to G-protein activation
The W3216.48 residue seals the top of the collapsed Na þ ion-binding pocket and along with the F3587.42 residue links the agonist peptide, bound closer to the extracellular surface, with residues in the lower part of NTSR1 that are implicated in conformational changes for GPCR activation
We describe structural, biochemical and pharmacological data of several NTSR1 mutants with either wild-type intracellular loop 3 (ICL3), or with most of ICL3 replaced by T4 lysozyme (T4L)
Summary
We analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Ga. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na þ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. We determined the structure of NTSR1 bound to NTS8–13 (Arg-Arg-Pro-Tyr-Ile-Leu) in an active-like conformation with six thermostabilizing mutations providing insight into the binding mode of a peptide agonist This receptor was unable to catalyse nucleotide exchange at the Ga subunit, indicating that some of the stabilizing mutations may have restricted the ability of NTSR1 to relate agonist binding to the activation of G protein. The double revertant NTSR1-EL (with E166 and L310) showed reduced activity in nucleotide exchange assays, but highlights the importance of E1663.49, of the highly conserved D/ERY motif, along with the neighbouring L3106.37 for G-protein activation (Fig. 1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
