Background and aims: The melanocortin-2-receptor accessory protein (MRAP) is essential for melanocortin-2-receptor (MC2R) function through receptor trafficking and signalling, enabling adrenal glucocorticoid synthesis in response to ACTH stimulation. Disabling mutations of MRAP result in life-threatening glucocorticoid deficiency, known as familial glucocorticoid deficiency type 2. MRAP has a single paralogue in the human genome, MRAP2. In vitro MRAP2 has a similar action to MRAP, facilitating MC2R function. However patients with FGD type 2 fail to be rescued by a functioning MRAP2 protein the aim of this study is to investigate why.
Methods: cAMP production in response to ACTH stimulation of HEK293 cells transfected with MC2R and MRAP/MRAP2/MRAP and MRAP2 was quantified using dual-reporter luciferase assays. In situ hybridisation (ISH) was used to localise adrenal MRAP, MRAP2 and MC2R mRNA expression. MRAP and MRAP2 adrenal expression was quantified using real-time qPCR.
Results: Higher concentrations of ACTH were required to induce cAMP production in HEK293 cells transfected with MC2R/MRAP2 compared with cells transfected with MC2R/MRAP (EC50 9.33e-007 vs 1.08e-009; P<0.0001). ISH demonstrated MRAP and MC2R expression in the adult rat zona fasciculata (ZF), (in cells capable of glucocorticoid synthesis), with highest concentrations in the undifferentiated zone (in cells incapable of glucocorticoid synthesis). MRAP2 was expressed at low levels throughout the adrenal cortex. Real-time qPCR of adrenal cDNA demonstrated a 20-fold greater expression of MRAP compared with MRAP2.
Conclusions: MRAP2 enables MC2R function only when stimulated with supraphysiological ACTH concentrations.
MRAP expression is 20-fold greater than MRAP2 expression in the human adrenal gland.
MRAP2 is expressed throughout the rat adrenal cortex. Its role in the adrenal gland remains unclear.
Adrenal MRAP and MC2R expression is confined to the ZF and highest expression is seen in the undifferentiated zone of the adult rat adrenal, suggesting ACTH may have a role in adrenal cell differentiation and migration.