Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides

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Abstract

Proadrenomedullin N-terminal 20 peptide (PAMP[1–20]/PAMP-20) and its truncated analog, PAMP[9–20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was “PAMP-12  cortistatin > PAMP-20”. These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca2+ mobilization, and [35S]guanosine 5′-(γ-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only Gαq but also Gαi, consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

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Materials and methods

Materials. Human PAMP-12 and PAMP-20 were purchased from the Peptide Institute (Osaka, Japan). Human cortistatin (CST-14) and BAM-22P were from Phoenix Pharmaceutical (Belmont, CA, USA) and Bachem (Bubendorf, Switzerland), respectively. 3-Isobutyl-1-methylxanthine (IBMX), pertussis toxin (PTX), and guanosine 5′-diphosphate (GDP) were from Sigma (St. Louis, MO, USA). Forskolin and methotrexate were from Wako (Osaka, Japan). [35S]guanosine 5′-(γ-thio)triphosphate ([35S]GTPγS) (1000 Ci/mmol) was

PAMP-12 specifically responds to MrgX2

Previous studies revealed that it is possible for a presumed PAMP receptor(s) to be a Gαi/o-protein-coupled receptor [16], [17], [18]. Therefore, based on the change in intracellular cAMP, we have searched for a PAMP-responsive receptor(s) from each type of orphan GPCR-expressing cell. By exploring these potential sources, we found that CHO cells stably expressing MrgX2 were specifically activated by PAMP-12 (Fig. 1A). MrgX2 has been classified as a member of the GPCR subfamily referred to as

Discussion

The proadrenomedullin gene products, PAMP-12 and PAMP-20, act as circulating hormones with hypotensive activities. Clinically, plasma levels of PAMP have been reported to increase in patients with a variety of diseases such as essential hypertension [28], chronic renal failure [29], congestive heart failure [30], and chronic glomerulonephritis [31]. This suggests that the vasophysiological function of PAMP relates to many diseases. Although clinical evidence indicates the importance of PAMP

Acknowledgments

We thank Dr. Shigekazu Nagata for providing pEF-BOS, and Ms. Mariko Isshiki, Ms. Akiko Wakita, and Mr. Hideki Miyamoto for excellent technical assistance.

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    Abbreviations: GPCR, G-protein-coupled receptor; PAMP, proadrenomedullin N-terminal peptide; AM, adrenomedullin; CST, cortistatin; Mrg, Mas-related gene; PTX, pertussis toxin; GTPγS, guanosine 5′-(γ-thio)triphosphate; DRG, dorsal root ganglia; EC50, median effective concentration.

    1

    These authors contributed equally to this work.

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