A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens  Vemuri B. Reddy, PhD, Thomas A. Graham, MD, PhD, Ehsan Azimi, MD, Ethan A. Lerner, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 6, Pages 1726-1728 (December 2017) DOI: 10.1016/j.jaci.2017.05.046 Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 SP activates MRGPRX2, MrgprA1, and MrgprB2 but not site-specific mutagenesis of Glu164 to Arg164 in MRGPRX2, and the equivalent residues in MrgprA1 and MrgprB2. HeLa cells were transfected with cDNAs encoding the mutant receptors. An HEK-293 cell line stably expressing the receptors was used for MRGPRX2, MrgprA1, and MrgprB2. Intracellular calcium [Ca2+]i was determined by ratiometric Fura-2 imaging after addition of SP as an indicator of receptor activation. Two other MRGPRX2 mutants, MRGPRX2T181A and MRGPRX2K251E, were generated as controls and SP activated these receptors in a manner similar to the native receptor (data not shown). Journal of Allergy and Clinical Immunology 2017 140, 1726-1728DOI: (10.1016/j.jaci.2017.05.046) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 SP binds to wild-type but not mutant Mrgprs. ELISA was used to quantify the binding of SP to wild-type receptors (MRGPRX2, MrgprA1, and MrgprB2) and mutated receptors (MRGPRX2E164R, MRGPRX2T181A, MRGPRX2K251E, MrgprA1N172R, and MrgprB2E171R). P values for **MRGPRX2 vs MRGPRX2E164R, ***MrgprA1 vs MrgprA1N172R, and **MrgprB2 vs MrgprB2E171R, are .0021, .0003, and .0016, respectively. P values greater than .05 are considered nonsignificant (ns). Journal of Allergy and Clinical Immunology 2017 140, 1726-1728DOI: (10.1016/j.jaci.2017.05.046) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions