Interactions between the Mas-Related Receptors MrgD and MrgE Alter Signalling and Trafficking of MrgD

Abstract

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to ␤-alanine but not L-alanine by elevating intracellular [Ca 2ϩ ], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to ␤-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of ␤-alanine to phosphorylate ERK1 and ERK2 as well as maintaining the capacity of ␤-alanine to elevate intracellular [Ca 2ϩ ], which was reduced in magnitude and slowed in response with increasing times of expression of rMrgD in isolation. Associated with these effects, the presence of rMrgE restricted ␤-alanine-induced internalization of rMrgD. This is the first report of heterodimeric interactions between members of the Mas-related gene (Mrg) receptor family and indicates that interactions between rMrgD and rMrgE modulate the function of rMrgD. Because the Mrg receptors are potential therapeutic targets in pain, these results suggest that efforts to understand the function and regulation of individual Mrg family receptors may require coexpression of relevant pairs.

Discovery and Characterization of Alamandine

“…The amino acid β-alanine has been reported to be an agonist of MrgD; however, the effective concentration of β-alanine in MrgD-transfected cells is considerably high (10 -3 to 10 -6 mol/L). 22,23 We first tested whether β-alanine and alamandine could interact in blood vessels. As shown in Figure 2F, β-alanine did not induce a direct relaxing effect in aortic rings.…”

Protean Agonism at the Dopamine D2 Receptor: (S)-3-(3-Hydroxyphenyl)-N-propylpiperidine Is an Agonist for Activation of Go1 but an Antagonist/Inverse Agonist for Gi1,Gi2, and Gi3

“…Although the fusion proteins described above have a major advantage in defining and ensuring the receptor-to-G protein ratio for each G protein, they are an inherently artificial system. To examine whether S-(Ϫ)-3-PPP would also behave as a protean agonist at the D2L receptor when regulating different G proteins in a system expressing separated receptor and G protein, we generated a series of HEK293 cell lines based on the Flp-In T-REx system (Ellis et al, 2006;Milasta et al, 2006). In these cell lines, the D2L receptor was expressed stably and constitutively, whereas the pertussis toxin-resistant cysteine to isoleucine forms of G␣ i1 , G␣ i2 , G␣ i3 , and G␣ o1 were cloned into the Flp-In locus, allowing their expression in an entirely inducible manner from the same single defined chromosomal locus by the addition of tetracycline.…”

MrgD Activation Inhibits KCNQ/M-Currents and Contributes to Enhanced Neuronal Excitability

“…MrgD is expressed across species from rodents to nonhuman primates and humans (Zhang et al, 2005). In addition, its expression is restricted to dorsal root ganglion (DRG) neurons (Dong et al, 2001;Shinohara et al, 2004;Milasta et al, 2006), specifically, nonpeptidergic, small-diameter, IB4 ϩ C-fiber neurons (Dong et al, 2001;Zylka et al, 2005). Together, these properties of MrgD provide for an experimentally tractable therapeutic target for pain.…”