Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions.
Monocyte chemotactic protein-1 (MCP-1) binds its G-protein-coupled seven transmembrane (TM) receptor, CCR2B, and causes infiltration of monocytes/macrophages into areas of injury, infection or inflammation. To identify functionally important amino acid residues in CCR2B, we made specific mutations of nine residues selected on the basis of conservation in chemokine receptors and located TM1 (Tyr(49)), TM2 (Leu(95)), TM3 (Thr(117) and Tyr(120)), and TM7 (Ala(286), Thr(290), Glu(291), and His(297)) and in the extracellular loop 3 (Glu(278)). MCP-1 binding was drastically affected only by mutations in TM7. Reversing the charge at Glu(291) (E291K) and at His(297) (H297D) prevented MCP binding although substitution with Ala at either site had little effect, suggesting that Glu(291) and His(297) probably stabilize TM7 by their ionic interaction. E291A elicited normal Ca(2+) influx. H297A, Y49F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca(2+) influx and elicited no adenylate cyclase inhibition at any MCP-1 concentration. MCP-1 treatment of HEK293 cells caused lamellipodia formation only when they expressed CCR2B. The mutants that showed no Ca(2+) influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation, but not Ca(2+) influx and adenylate cyclase inhibition, is necessary for chemotaxis.
Gavrilin
Mikhail A
MA
Davis Heart and Lung Reseach Institute, The Ohio State University, Columbus, OH 43210, USA. gavrilin.1@osu.edu
Gulina
Irina V
IV
Kawano
Tomonori
T
Dragan
Sofya
S
Chakravarti
Leena
L
Kolattukudy
Pappachan E
PE
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D018932:Chemokine CCL2 / Q000378:metabolism* / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000235:genetics; D003599:Cytoskeleton / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011554:Pseudopodia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D000067956:Adenylyl Cyclase Inhibitors; D000596:Amino Acids; C515334:CCR2 protein, human; D018932:Chemokine CCL2; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D000262:Adenylyl Cyclases; D002118:Calcium