Third extracellular loop (EC3)-N terminus interaction is important for seven-transmembrane domain receptor function:...

Area of Science:

• Biochemistry and Molecular Biology
• Cellular Signaling
• Pharmacology

Background:

• Seven-transmembrane domain (7TM) receptors possess conserved cysteine pairs in the third extracellular loop (EC3) and N terminus.
• The functional significance of these EC3-N terminus cysteine pairs in 7TM receptor conformation and signaling remains largely undefined.

Purpose of the Study:

• To investigate the role of EC3-N terminus cysteine pairs in G protein-coupled receptor (GPCR) activation and signaling.
• To explore how disrupting conserved cysteine pairs in CXCR4 and engineering them into C5aR impacts receptor function.

Main Methods:

• Site-directed mutagenesis was employed to disrupt or engineer cysteine pairs in CXCR4 and C5aR.
• Mutated receptors were expressed in engineered yeast and COS-7 cells for functional analysis.
• Receptor activation and G protein signaling were assessed in response to specific ligands.

Main Results:

• Mutation of the cysteine pair to serine in constitutively active CXCR4 abrogated receptor activation.
• Mutation to aromatic or salt bridge pairs in CXCR4 rescued or retained activation by CXCL12.
• Engineering a cysteine pair into C5aR restrained constitutive signaling without affecting C5a-induced activation, highlighting a new microswitch region.

Conclusions:

• EC3-N terminus cysteine pairs play a novel and critical role in regulating GPCR conformation and G protein activation.
• These cysteine pairs act as modulators of both constitutive and ligand-induced signaling pathways in 7TM receptors.