The Meaning of Mas

In the early 1980s, in their search for systems that import proteins into mitochondria, Yaffe and Schatz identified a mutant in the yeast Saccharomyces cerevisiae, mas1 (for mitochondrial assembly 1), that accumulates mitochondrial precursor proteins (Table 1).¹ In 1988 they cloned and sequenced the wild type yeast MAS1 gene (systematic name: YLR163C), which encodes the catalytic subunit of the mitochondrial processing protease, a component of the mitochondrial import pathway and essential for cell viability.² Later, homologs of this gene were found in other eukaryotes including humans, in which the gene was called PMPCB (Peptidase, mitochondrial processing beta subunit).³ Around the same time, in 1986, a new gene was isolated from DNA of a human epidermoid carcinoma cell line, identified as a proto-oncogene, and named MAS.⁴ Initially the function of the MAS protein was unknown and it was only in the early 2000s that it was identified as the G protein-coupled receptor (GPCR) through which angiotensin (Ang)-(1-7) signals. Unfortunately, the MAS gene was later renamed by the HUGO Human Gene Nomenclature Committee to MAS1 (Full name: MAS1 proto-oncogene, G protein-coupled receptor) and also in mouse, rat and all other tetrapods it got the new name Mas1. Fortunately, MAS is still an accepted alias of the MAS1 proto-oncogene protein and we will use this name in the following to distinguish it from the yeast Mas1 protein. We would also like to suggest that the name MAS should be used in future publications. There is no homolog of MAS in any clade outside tetrapods.⁵ However, several homologous genes were discovered in each tetrapod species and the name MAS was given to this new family of receptors, the Mas-related GPCRs (Mrgprs).^5,6

Table 1.

Comparison of different MAS1 genes. The rules for the nomenclature of genes and proteins differ between organisms. In humans (https://www.genenames.org/about/guidelines), both are in upper case letters and gene symbols are italicized. In rats and mice (http://www.informatics.jax.org/mgihome/nomen/gene.shtml#pon) only protein symbols are in uppercase letters and gene symbols have only an initial uppercase letter and are also italicized. In yeast, gene symbols are in upper case italicized letters and proteins are referred to by the relevant gene symbol, non-italic, only initial letter uppercase and with the suffix ‘p’ (which can be omitted when the context reveals that the protein is meant) (http://seq.yeastgenome.org/nomenclature-conventions). In bacteria, gene symbols are in all lower case letters and italicized, protein names are non-italic with only the first letter in upper case.⁵⁴

Organism	Gene	Protein	Aliases	Full name	Database IDs	Molecular mass	Function
Human	MAS1	MAS1	MAS	MAS1 Proto-Oncogene, G Protein-Coupled Receptor	NCBI: 4142 HGNC: 6899	37 kDA	G protein-coupled receptor, Angiotensin-(1-7) receptor
Mouse	Mas1	MAS1	Mas MasR Mas-1	MAS1 oncogene	NCBI: 17171 MGI: 96918	37 kDA
Yeast	MAS1	Mas1p	Mas1 YLR163C MIF	Mitochondrial-ASsembly protein 1	NCBI: 850860 SGD: S000004153	51 kDA	Catalytic subunit of the mitochondrial processing protease
Strepto-myces sp. W007	mas1 (SPW_1544)	Mas1	H0B8D4	MArine Streptomyces 1	Uniprot: H0B8D4	31 kDa	Putative secreted thermostable lipase

The duplication in nomenclature (Table 1) has unfortunately resulted in some misunderstandings and confusion in the Ang field, because there are some papers that attribute Ang-(1-7) effects to yeast mitochondrial assembly protein 1, Mas1.^7–10 This may be especially important in the context of interpretation of results and consideration of tools used to interrogate mammalian MAS, because it is likely that in some studies antibodies to the yeast Mas1 protein rather than to the GPCR MAS may have been used erroneously. To further add to the complexity, the molecular size of the yeast Mas1 protein (~ 50 kDa) is not that dissimilar to that of human MAS (~ 40 kDa) and antibodies against MAS, which we tested, were nonspecific.¹¹ The confusion was additionally increased in 2016, when a putative thermostable lipase from a marine Streptomyces species was also named Mas1 (Table 1)¹². However, at least until now this protein has not been confused with MAS.

The aim of this brief review is to highlight the importance of discriminating between the different ‘Mas1’ proteins and to ensure that the GPCR MAS is indeed the protein of interest when examining Ang-(1-7) (patho)physiological actions. Here we provide a historical overview of MAS and describe the origin of the name and how its functions have been unravelled.

Discovery of MAS as a Proto-oncogene

The MAS gene was first identified in 1986 using an assay for human oncogenes based on their ability to induce tumorigenicity of NIH 3T3 cells in nude mice.^13,14 Briefly, NIH 3T3 cells were cotransfected with DNA purified from a human tumor along with a G418 selectable marker. After selection and growth in culture, the G418 resistant cells were injected into nude mice. Several weeks later, DNA from tumors that formed in the mice was purified. The human DNA isolated from one of these tumors contained the MAS gene. The name MAS is an abbreviation of the last name (Massey) of the person who donated the human tumor from which the MAS gene was derived. This gene was cloned and shown to possess the ability to induce NIH 3T3 cells to form foci of transformed cells in culture and to form tumors in nude mice.⁴ Therefore MAS was called a proto-oncogene. However, MAS likely did not contribute to the formation of the human tumor since the gene did not appear to be rearranged or mutated in the original human tumor DNA; rather, the transforming potential of MAS in NIH 3T3 cells appeared to be activated by DNA rearrangement and/or amplification during transfection into NIH 3T3 cells.^4,15 Moreover recent findings have suggested that MAS-activation by Ang-(1-7) could actually be a therapeutic target against tumors and has been suggested as a putative anti-cancer treatment.¹⁶

MAS as Angiotensin II Receptor?

Already at the time of its discovery, the DNA sequence of the MAS gene was determined and shown to encode a protein with a seven-transmembrane domain structure similar to that of GPCR.⁴ Despite the fact that only one protein is encoded by the gene, we recently demonstrated that the mouse Mas gene with 4 promoters and 12 exons generates at least 12 different mRNAs by alternative splicing at the 5’ untranslated region and is thereby the most complex gene of all GPCRs¹⁷. In order to define its ligand, Jackson et al.¹⁸ expressed MAS in Xenopus oocytes and in a mammalian cell line. Oocytes exhibited a dose-dependent induction of an inward current in response to angiotensin (Ang) I, II, and III, and in transfected cells Ang II and III led to intracellular Ca²⁺ release and to the initiation of DNA synthesis. Based on these results MAS was suggested to be a functional Ang II receptor. However, whereas several follow-up studies supported this assumption^19–22, Ambroz et al.²³ showed that the Ca²⁺ release after Ang II treatment was only observed in MAS-transfected cells additionally expressing endogenous Ang II receptors. Cloning of the real Ang II receptor, AT₁, in 1991^24,25 and the discovery of a direct interaction between MAS and AT₁ in 2005,^26,27 partly explained the original observations of Jackson et al.¹⁸ in Xenopus oocytes and revealed that MAS is not an Ang II receptor per se, but modulates AT₁ signaling.

Mas as Imprinted Gene?

In 1994, Mas was reported to be maternally imprinted in mice²⁸ and in human breast tissue,²⁹ i.e., one of the two parental Mas alleles was epigenetically silenced. The Mas gene is located in close proximity to the imprinted Igf2r gene in the human and mouse genomes.^30,31 Imprinting of this chromosomal area is regulated by an intronic control element starting the transcription of the long noncoding RNA, Airn (Antisense Igf2r RNA Noncoding). The transcribed antisense RNA overlaps (and silences) the Igf2r promoter and partially the Mas gene.^32,33 Using Mas-deficient mice³⁴ we could show that Mas is biallelically expressed.³⁵ Since Villar and Pedersen²⁸ and Miller et al.,²⁹ used RT-PCR assays which lack strand selectivity to discover imprinting of Mas it is very likely that they detected Airn as maternally imprinted RNA and not the Mas transcript. Thus, Airn but not Mas is monoallelically expressed in mouse and man.

MAS as Angiotensin-(1-7) Receptor

The first evidence for a receptor for Ang-(1-7) distinct from the Ang II receptors came from the observation that Ang-(1-7) was equipotent to Ang II for vasopressin release from hypothalamus-neurohypophyseal explants,³⁶ but in contrast to Ang II had no effect on drinking behavior.³⁷ Moreover, Ang-(1-7) was reported to exert vasodilatory effects by releasing NO resulting in a blood pressure decrease.³⁸ This and other actions of Ang-(1-7), which all opposed the effects of Ang II, further supported that Ang-(1-7) mediates its effects through a novel non-AT₁/AT₂ receptor subtype. The final proof for the existence of a specific receptor for the peptide was the discovery of a selective antagonist for Ang-(1-7) in 1994.^39,40

Yet, it was only in 2003 that more definitive evidence for a specific binding site for Ang-(1-7) was demonstrated with the finding that MAS is a receptor for the heptapeptide.⁴¹ In that study, specific binding of ¹²⁵I-Ang-(1-7) to Mas-transfected cells was reported. Moreover, the specific binding of ¹²⁵I-Ang-(1-7) but not of ¹²⁵I-Ang II or ¹²⁵I-Ang IV to kidney sections, was abolished by genetic deletion of Mas. In addition, Mas-deficient mice completely lack the antidiuretic action of Ang-(1-7) after an acute water load and Mas-deficient aortas lost their Ang-(1-7)-induced relaxation response. These findings provided the first clear molecular basis for the physiological actions of this biologically active peptide. At this point an orphan receptor met an orphan peptide filling an important gap in our understanding of the renin-angiotensin system. Further support for these findings was obtained in different laboratories. In 2005, Tallant et al.⁴² showed that transfection of cultured myocytes with an antisense oligonucleotide to Mas blocked the Ang-(1-7)-mediated inhibition of serum-stimulated MAPK activation, whereas a sense oligonucleotide was ineffective. Ang-(1-7) was found to stimulate NO release and eNOS activation in endothelial cells and these effects were blocked by the specific MAS-antagonist, A-779^43,44. In addition, Mas-deficiency abolishes all the known cardiovascular effects of Ang-(1-7).⁴⁵ Indeed, in most instances genetic deletion of Mas causes alterations opposed to those produced by treatment with Ang-(1-7).

Nevertheless, there are recent reports that Ang-(1-7) has no effect on MAS-transfected cells but exerts biased agonism or even antagonism at the AT₁ receptor.^46–48 Moreover, using other MAS agonists (NPFF and AR234960) and inverse agonists (AR244555) biased signaling of MAS itself was described.⁴⁹ Heteromeric interactions of MAS with AT₁, AT₂, bradykinin B2 and endothelin B receptors further complicate this issue.^{26,27,50–52} Therefore future studies need to clarify the relationship between MAS and Ang-(1-7) which may depend on the specific cell types and their expression of other GPCRs.⁵³

Conclusions

In conclusion, this brief review highlights some important points related to some misconceptions and confusions regarding the nomenclature of MAS and its functions (Table), especially in the context of cardiovascular pathophysiology. We suggest that the original name of “Mas” be used for the GPCR.

Important take home messages.

1. The MAS1 gene in yeast codes for Mas1p (mitochondrial assembly protein 1) a protease essential for protein import into mitochondria and homologous to the human PMPCB gene.

2. MAS1 or MAS in tetrapods is a G protein-coupled receptor for Ang-(1-7), but not for Ang II.

3. Yeast Mas1 protein has a molecular size of 50-52 kDa, while mammalian MAS has a molecular size of 37-40 kDa.

4. When probing for MAS1 or MAS in the context of Ang-(1-7) biology, ensure the correct primers and antibodies are used to assess expression of mRNA and protein respectively. It should be noted though that currently the authors are unaware of commercially available antibodies that specifically detect MAS at physiological expression levels. However, we demonstrated that the following primer pair is suitable to quantify human MAS mRNA by qPCR and may also be used in mice: 5’-GCTACAACACGGGCCTCTATCTG-3’; 5’-TACTCCATGGTGGTCACCAAGC-3’, fragment length 160 bp.

5. The mouse Mas gene is not imprinted.

6. The MAS gene is a proto-oncogene, but has not yet been shown to cause a human tumor.

7. Ang-(1-7)/MAS mediates effects that oppose actions of Ang II/AT₁.

8. MAS interacts with other G protein-coupled receptors.