Identification of Regions Involved in the Physical Interaction between Melanocortin Receptor Accessory Protein 2 and Prokineticin Receptor 2

Abstract

Melanocortin Receptor Accessory Protein 2 (MRAP2) modulates the trafficking and signal transduction of several G-protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, such as Prokineticin receptors (PKRs). They bind the endogenous ligand prokineticin 2 (PK2), a novel adipokine that has an anorexic effect and modulates thermoregulation and energy homeostasis. In the present work, we used biochemical techniques to analyze the mechanism of interaction of MRAP2 with PKR2 and we identified the specific amino acid regions involved in the complex formation. Our results indicate that MRAP2 likely binds to the N-terminal region of PKR2, preventing glycosylation and consequently the correct receptor localization. We also identified a C-terminal region of MRAP2 that is critical for the interaction with PKR2. Consequently, we analyzed the role of the prokineticin transduction system in the regulation of MRAP2 expression in tissues involved in the control of food intake: at the central level, in hypothalamic explants, and at the peripheral level, in adipocytes. We demonstrated the modulation of MRAP2 expression by the prokineticin transduction system.

Arginine 125 Is an Essential Residue for the Function of MRAP2

“…The C-terminal MRAP2 domain is involved in PKR2 localization and activation. Recently, we identified a region from asparagine at position 77 to alanine at position 131 involved in specific interaction with the extracellular N-terminal sequence of PKR2 [ 5 ]. The arginine residue at position 125 is extremely conserved in primates and replaced by histidine in rodents ( Figure 1 A).…”

“…In CHO cells stably transfected with PKR2, PK2 is able to activate ERK and this activation is abrogated by pretreatment with the G αi inhibitor pertussis toxin [ 6 ]. Moreover, the effect of PK2 is significantly reduced in the presence of MRAP2 [ 5 ]. We therefore, examined ERK and STAT3 phosphorylation after PK2 incubation of CHO-PKR2 cells in the presence or absence of the MRPA2 mutants R125H and R125C.…”

“…The C-terminal region of MRAP2 encoded by the fourth exon (MRAP2-CT) retains the ability to dimerize, and the protein migrates as a dimer even under denaturing conditions, as described previously [ 5 ]. We performed a GST pull-down experiment to determine the ability of mouse MRAP2 and the MRAP2 mutants to form dimers.…”

“…MRAP2 has the ability to form a unique topology in eukaryotes characterized by a dual-oriented dimer structure. Our results defined the minimal C-terminal domain region of MRAP2 as sufficient to ensure dimer formation [ 5 ]. Other MRAP2 regions were identified as important for MRAP2 dimer formation, particularly the N-terminal region and the transmembrane domain [ 18 , 19 ].…”

“…MRAP2 interacts with several G protein-coupled receptors (GPCRs), such as melanocortin, ghrelin, and orexin receptors, and influences their expression, localization, signaling, and internalization. Recently, MRAP2 has been shown to increase food intake by inhibiting prokineticin receptors (PKRs) [ 3 , 4 , 5 ]. PKRs are GPCRs expressed both centrally and peripherally.…”