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Localisation of the melanocortin-2-receptor and its accessory proteins in the developing and adult adrenal gland.

Gorrigan RJ, Guasti L, King P, Clark AJ, Chan LF - J. Mol. Endocrinol. (2011)

Bottom Line: In this study on the rat adrenal gland, we demonstrate that while MRAP and MC2R are highly expressed in the zona fasciculata, MRAP2 is expressed throughout the adrenal cortex in low quantities.The MC2R/MRAP2 complex requires much higher concentrations of ACTH to activate compared with the MC2R/MRAP complex.Interestingly, expression of MC2R and MRAP in the undifferentiated zone would support the notion that ACTH may play an important role in adrenal cell differentiation and maintenance.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Centre for Endocrinology, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, London EC1M 6BQ, UK.

ABSTRACT
The melanocortin-2-receptor (MC2R)/MC2R accessory protein (MRAP) complex is critical to the production of glucocorticoids from the adrenal cortex. Inactivating mutations in either MC2R or MRAP result in the clinical condition familial glucocorticoid deficiency. The localisation of MC2R together with MRAP within the adrenal gland has not previously been reported. Furthermore, MRAP2, a paralogue of MRAP, has been shown in vitro to have a similar function to MRAP, facilitating MC2R trafficking and responsiveness to ACTH. Despite similar MC2R accessory functions, in vivo, patients with inactivating mutations of MRAP fail to be rescued by a functioning MRAP2 gene, suggesting differences in adrenal expression, localisation and/or function between the two MRAPs. In this study on the rat adrenal gland, we demonstrate that while MRAP and MC2R are highly expressed in the zona fasciculata, MRAP2 is expressed throughout the adrenal cortex in low quantities. In the developing adrenal gland, both MRAP and MRAP2 are equally well expressed. The MC2R/MRAP2 complex requires much higher concentrations of ACTH to activate compared with the MC2R/MRAP complex. Interestingly, expression of MC2R and MRAP in the undifferentiated zone would support the notion that ACTH may play an important role in adrenal cell differentiation and maintenance.

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In situ hybridisation showing localisation of Mrap, Mrap2 and Mc2r in foetal and adult Wistar rat adrenal glands. (A–G) Foetal Wistar rat adrenal glands (embryonic age 17·5 days); (H–N) adult male Wistar rat adrenal glands. (A and B) Low-magnification images of foetal rat adrenal glands incubated with Mrap antisense and sense (S) riboprobes. Mrap is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (C and D) 10× magnified images of foetal rat adrenal glands. No Mrap staining in the adrenal capsule (C) or subcapsular zone (indicated by T). (E and F) Foetal rat adrenal glands incubated with Mrap2 antisense and sense (S) riboprobes. Mrap2 is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (G) Combined Mrap ISH and CYP11B1 immunohistochemistry. Nuclei are stained with DAPI. Cells positive for Mrap also express CYP11B1, suggesting these cells are capable of glucocorticoid synthesis and are destined to localise to the zona fasciculata postnatally. (H–K) Mc2r and Mrap are present in the zona fasciculata (ZF), with relative sparing of the capsule, zona glomerulosa (ZG) and medulla (M). Expression was strongest at the periphery, with a gradient reduction towards the medulla. The sense probes resulted in no specific staining (h, j). (L) Combined Mc2r ISH and CYP11B1 immunohistochemistry. Mc2r is expressed in the ZF, in cells co-expressing CYP11B1. Mc2r is also expressed highly in the undifferentiated zone, in cells which lack CYP11B1 expression. (M and N) Mrap2 is expressed at low levels throughout the adult rat adrenal gland. The sense probe resulted in no specific staining (N).
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fig1: In situ hybridisation showing localisation of Mrap, Mrap2 and Mc2r in foetal and adult Wistar rat adrenal glands. (A–G) Foetal Wistar rat adrenal glands (embryonic age 17·5 days); (H–N) adult male Wistar rat adrenal glands. (A and B) Low-magnification images of foetal rat adrenal glands incubated with Mrap antisense and sense (S) riboprobes. Mrap is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (C and D) 10× magnified images of foetal rat adrenal glands. No Mrap staining in the adrenal capsule (C) or subcapsular zone (indicated by T). (E and F) Foetal rat adrenal glands incubated with Mrap2 antisense and sense (S) riboprobes. Mrap2 is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (G) Combined Mrap ISH and CYP11B1 immunohistochemistry. Nuclei are stained with DAPI. Cells positive for Mrap also express CYP11B1, suggesting these cells are capable of glucocorticoid synthesis and are destined to localise to the zona fasciculata postnatally. (H–K) Mc2r and Mrap are present in the zona fasciculata (ZF), with relative sparing of the capsule, zona glomerulosa (ZG) and medulla (M). Expression was strongest at the periphery, with a gradient reduction towards the medulla. The sense probes resulted in no specific staining (h, j). (L) Combined Mc2r ISH and CYP11B1 immunohistochemistry. Mc2r is expressed in the ZF, in cells co-expressing CYP11B1. Mc2r is also expressed highly in the undifferentiated zone, in cells which lack CYP11B1 expression. (M and N) Mrap2 is expressed at low levels throughout the adult rat adrenal gland. The sense probe resulted in no specific staining (N).

Mentions: ISH was performed to visualise the localisation of Mrap, Mrap2 and Mc2r mRNA expression within the adrenal gland (Fig. 1). Mrap and Mrap2 were readily detectable specifically in the adrenal gland during development (Fig. 1A–F). Mrap and Mrap2 NR-ISH was performed in combination with CYP11B1 IHC, which demonstrated that cortical, but not medullary cells, expressed Mc2r accessory proteins (Fig. 1G–G″).


Localisation of the melanocortin-2-receptor and its accessory proteins in the developing and adult adrenal gland.

Gorrigan RJ, Guasti L, King P, Clark AJ, Chan LF - J. Mol. Endocrinol. (2011)

In situ hybridisation showing localisation of Mrap, Mrap2 and Mc2r in foetal and adult Wistar rat adrenal glands. (A–G) Foetal Wistar rat adrenal glands (embryonic age 17·5 days); (H–N) adult male Wistar rat adrenal glands. (A and B) Low-magnification images of foetal rat adrenal glands incubated with Mrap antisense and sense (S) riboprobes. Mrap is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (C and D) 10× magnified images of foetal rat adrenal glands. No Mrap staining in the adrenal capsule (C) or subcapsular zone (indicated by T). (E and F) Foetal rat adrenal glands incubated with Mrap2 antisense and sense (S) riboprobes. Mrap2 is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (G) Combined Mrap ISH and CYP11B1 immunohistochemistry. Nuclei are stained with DAPI. Cells positive for Mrap also express CYP11B1, suggesting these cells are capable of glucocorticoid synthesis and are destined to localise to the zona fasciculata postnatally. (H–K) Mc2r and Mrap are present in the zona fasciculata (ZF), with relative sparing of the capsule, zona glomerulosa (ZG) and medulla (M). Expression was strongest at the periphery, with a gradient reduction towards the medulla. The sense probes resulted in no specific staining (h, j). (L) Combined Mc2r ISH and CYP11B1 immunohistochemistry. Mc2r is expressed in the ZF, in cells co-expressing CYP11B1. Mc2r is also expressed highly in the undifferentiated zone, in cells which lack CYP11B1 expression. (M and N) Mrap2 is expressed at low levels throughout the adult rat adrenal gland. The sense probe resulted in no specific staining (N).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: In situ hybridisation showing localisation of Mrap, Mrap2 and Mc2r in foetal and adult Wistar rat adrenal glands. (A–G) Foetal Wistar rat adrenal glands (embryonic age 17·5 days); (H–N) adult male Wistar rat adrenal glands. (A and B) Low-magnification images of foetal rat adrenal glands incubated with Mrap antisense and sense (S) riboprobes. Mrap is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (C and D) 10× magnified images of foetal rat adrenal glands. No Mrap staining in the adrenal capsule (C) or subcapsular zone (indicated by T). (E and F) Foetal rat adrenal glands incubated with Mrap2 antisense and sense (S) riboprobes. Mrap2 is present throughout the foetal rat adrenal gland. The sense probe resulted in no specific staining. (G) Combined Mrap ISH and CYP11B1 immunohistochemistry. Nuclei are stained with DAPI. Cells positive for Mrap also express CYP11B1, suggesting these cells are capable of glucocorticoid synthesis and are destined to localise to the zona fasciculata postnatally. (H–K) Mc2r and Mrap are present in the zona fasciculata (ZF), with relative sparing of the capsule, zona glomerulosa (ZG) and medulla (M). Expression was strongest at the periphery, with a gradient reduction towards the medulla. The sense probes resulted in no specific staining (h, j). (L) Combined Mc2r ISH and CYP11B1 immunohistochemistry. Mc2r is expressed in the ZF, in cells co-expressing CYP11B1. Mc2r is also expressed highly in the undifferentiated zone, in cells which lack CYP11B1 expression. (M and N) Mrap2 is expressed at low levels throughout the adult rat adrenal gland. The sense probe resulted in no specific staining (N).
Mentions: ISH was performed to visualise the localisation of Mrap, Mrap2 and Mc2r mRNA expression within the adrenal gland (Fig. 1). Mrap and Mrap2 were readily detectable specifically in the adrenal gland during development (Fig. 1A–F). Mrap and Mrap2 NR-ISH was performed in combination with CYP11B1 IHC, which demonstrated that cortical, but not medullary cells, expressed Mc2r accessory proteins (Fig. 1G–G″).

Bottom Line: In this study on the rat adrenal gland, we demonstrate that while MRAP and MC2R are highly expressed in the zona fasciculata, MRAP2 is expressed throughout the adrenal cortex in low quantities.The MC2R/MRAP2 complex requires much higher concentrations of ACTH to activate compared with the MC2R/MRAP complex.Interestingly, expression of MC2R and MRAP in the undifferentiated zone would support the notion that ACTH may play an important role in adrenal cell differentiation and maintenance.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Centre for Endocrinology, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, London EC1M 6BQ, UK.

ABSTRACT
The melanocortin-2-receptor (MC2R)/MC2R accessory protein (MRAP) complex is critical to the production of glucocorticoids from the adrenal cortex. Inactivating mutations in either MC2R or MRAP result in the clinical condition familial glucocorticoid deficiency. The localisation of MC2R together with MRAP within the adrenal gland has not previously been reported. Furthermore, MRAP2, a paralogue of MRAP, has been shown in vitro to have a similar function to MRAP, facilitating MC2R trafficking and responsiveness to ACTH. Despite similar MC2R accessory functions, in vivo, patients with inactivating mutations of MRAP fail to be rescued by a functioning MRAP2 gene, suggesting differences in adrenal expression, localisation and/or function between the two MRAPs. In this study on the rat adrenal gland, we demonstrate that while MRAP and MC2R are highly expressed in the zona fasciculata, MRAP2 is expressed throughout the adrenal cortex in low quantities. In the developing adrenal gland, both MRAP and MRAP2 are equally well expressed. The MC2R/MRAP2 complex requires much higher concentrations of ACTH to activate compared with the MC2R/MRAP complex. Interestingly, expression of MC2R and MRAP in the undifferentiated zone would support the notion that ACTH may play an important role in adrenal cell differentiation and maintenance.

Show MeSH
Related in: MedlinePlus