Cysteine at Position 217 in the Intracellular Loop 1 Plays a Critical Role in Human PTH Receptor Type 1 Membrane Translocation and Function

Abstract

PTHR1 mutants lacking endogenous cysteines in transmembrane and intracellular domains were generated. Mutant receptors were tested for their biological activities and mRNA and cell surface expression levels. C217 in intracellular loop 1 was determined to play a critical role in cell surface translocation and function of the receptor.Introduction: Elucidating the role of different domains of PTH receptor 1 (PTHR1) is essential for understanding the mechanism of ligand-receptor interactions. Here we present a study directed at determining the importance of cysteine residues present in the intracellular and transmembrane (TM) domains of the receptor. Materials and Methods: Mutant receptors were generated by site-directed mutagenesis. Biological activities were characterized by adenylyl cyclase and competition binding assays. RT-PCR, ELISA, and immunofluorescence microscopy were carried out to determine receptor mRNA and protein expression levels. Results: Mutations C460L and C462L in TM7, C568L in the C-terminal intracellular domain of the receptor, and removal of C397 in intracellular loop (ICL)3 by insertion of cleavage sites for Factor Xa did not affect binding affinity of PTH or agonist-induced adenylyl cyclase activity, although maximal responses (IC max and EC max ) were decreased. However, mutations C217L in ICL1 or both C217L and C568L simultaneously resulted in a decrease in binding and loss of adenylyl cyclase activity. RT-PCR results showed that the observed changes in binding and activity were not caused by changes in mRNA expression. Next, we determined cell surface and total expression of the wildtype and mutant receptors by ELISA. We found that mutations of C460/C462 to L moderately decreased transfer of receptors to the cell surface. However, mutation of C217 to L in the ICL1 drastically reduced cell surface expression. Immunofluorescence and confocal microscopy studies confirmed reduced cell surface expression of receptors containing the C217L mutation. Similar results were obtained when replacing C217 and C460/C462 of the receptor with A instead of L. Conclusions: Our studies indicate that the cysteine at position 217 in ICL1 plays a critical role in translocation to the cell surface and biological function of PTHR1.

PTH and PTH Antagonist Induce Different Conformational Changes in the PTHR1 Receptor

“…The wtPTHR1 was cloned previously into pcDNA 3.1 (16) . It was used as a template to insert the c‐myc epitope sequence (EQKLISEEDL) and FXa cleavage sites ([IEGR]2) as described previously (17) . The resulting PTHR1 construct is referred to as XM‐PTHR1 throughout this paper.…”

The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activation

“…In the inactive GCCR (5EE7) and GLP-1R (5VEW), it also H-bonds to a water molecule in the cleft between H8 and ICL1. Alanine mutation of C 2.44b in the PTH1 receptor results in loss of receptor expression (57), further suggestive of a role in all class B1 GPCRs. In the GCGR where the 2.45b is the shorter threonine a bridging water molecule allows a hydrogen bond between the backbone amides of 2.45b, 2,46b and the phenolic hydroxyl of Y248 3.53b (Figure 9H).…”

Oxidation inhibits PTH receptor signaling and trafficking

“…The high predisposition of ROS to affect cysteine residues is especially relevant given the role of GPCR and G-protein cysteine residues in the formation of intra- and inter-molecular disulfide bridges and receptor complexes, formation of ligand binding domains, and stabilization of protein conformations through modifications such as palmitoylation and prenylation, which facilitate downstream signal transduction [31]. PTHR harbors eight extracellular that have been shown to be essential for receptor function [32], and five additional intracellular cysteines distributed in loops 1 and 3, transmembrane domain 7, and the C-terminal intracellular tail.Cystein-217 in intracellular loop 1 is necessary for adenylyl cyclase/cAMP signaling [33]. It is possible though, that oxidation of cysteines in PTHR changes conformation of the receptor or modifies binding domains resulting in reduced interactions with signaling partners.…”