Third extracellular loop (EC3)-N terminus interaction is important for seven-transmembrane domain receptor function: implications for an activation microswitch region.
The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined by three intra- and three extracellular loops, whose local conformations are important in receptor signaling. Many 7TM receptors contain a cysteine residue in the third extracellular loop (EC3) and a complementary cysteine residue on the N terminus. The functional role of such EC3-N terminus conserved cysteine pairs remains unclear. This study explores the role of the EC3-N terminus cysteine pairs on receptor conformation and G protein activation by disrupting them in the chemokine receptor CXCR4, while engineering a novel EC3-N terminus cysteine pair into the complement factor 5a receptor (C5aR), a chemo attractant receptor that lacks it. Mutated CXCR4 and C5aRs were expressed in engineered yeast. Mutation of the cysteine pair with the serine pair (C28S/C274S) in constitutively active mutant CXCR4 abrogated the receptor activation, whereas mutation with the aromatic pair (C28F-C274F) or the salt bridge pair (C28R/C274E), respectively, rescued or retained the receptor activation in response to CXCL12. In this context, the cysteine pair (Cys(30) and Cys(272)) engineered into the EC3-N terminus (Ser(30) and Ser(272)) of a novel constitutively active mutant of C5aR restrained the constitutive signaling without affecting the C5a-induced activation. Further mutational studies demonstrated a previously unappreciated role for Ser(272) on EC3 of C5aR and its interaction with the N terminus, thus defining a new microswitch region within the C5aR. Similar results were obtained with mutated CXCR4 and C5aRs expressed in COS-7 cells. These studies demonstrate a novel role of the EC3-N terminus cysteine pairs in G protein-coupled receptor activation and signaling.
Rana
Soumendra
S
Division of Metabolism, Endocrinology, and Lipid Research, Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Baranski
Thomas J
TJ
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D009154:Mutation; D017433:Protein Structure, Secondary; D044087:Receptor, Anaphylatoxin C5a; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
C488033:C5AR1 protein, human; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D044087:Receptor, Anaphylatoxin C5a; D019718:Receptors, CXCR4; D011951:Receptors, Complement; D019204:GTP-Binding Proteins