Expression and function of novel members of the MS4A family
Creators: name: Leffler, Melanie
Publisher: name: The Australian National University
Year: 2010
Subjects: subject: QH450.L44 2010
subject: Cell interaction
subject: Gene expression
subject: Genetics
subject: FOS: Biological sciences

description: The MS4A (membrane-spanning four-domains, subfamily A) family encompasses a group of genes (MS4A1 to MS4A15 and TMEM176A and B) primarily clustered to chromosome 11q12-13. To date, the majority of MS4A family members have been reported to be expressed in cells of haematopoietic origin and to function in multimeric complexes that regulate cell activation and proliferation. The pan B cell marker, CD20 (MS4A1), was the first isolated MS4A molecule and has been shown to interact with the B cell receptor (BCR) at the cell surface to regulate BCR-dependent cytoplasmic calcium mobilisation and signalling pathways, ultimately playing a role in cell survival and proliferation. Fc{u03B5}RI{u03B2} (MS4A2) shares high amino acid sequence identity to CD20, is expressed in mast cells and basophils as part of the high affinity IgE receptor complex (Fc{u03B5}RI) and regulates IgE-mediated signalling pathways. The main role of Fc{u03B5}RI{u03B2} is in the recruitment of src-family protein tyrosine kinase, lyn, ultimately leading to enhancement of down-stream effector functions mediated through IgE-antigen binding of Fc{u03B5}RI, such as mast cell degranulation. Fc{u03B5}RI{u03B2} has also been proposed as a candidate gene for an atopy susceptibility locus localised at chromosome 11q13 although no profound phenotypic differences have been identified between linked polymorphisms and the wild-type form of Fc{u03B5}RI{u03B2}. A limited number of other MS4A family members have been functionally characterised although far less extensively. These include the perinuclear-expressed HTm4 (MS4A3) that is involved in haematopoietic cell cycling and MS4A12 that has been shown to play a regulatory role in colon epithelial cell cycling and store operate calcium channel (SOCC) opening in an epidermal growth factor receptor (EGFR)-dependent manner. Beyond these reports, little is known about the majority of the MS4A family members and fundamental information is lacking including amino acid sequence characteristics, cellular and subcellular expression distribution and functional roles. In order to address some of these deficiencies in our understanding of the MS4A family, the aim of this thesis was to further characterise the expression distribution of MS4A family members, identify novel homologous genes and investigate the function of one specific family member, MS4A8B. The analysis of sequence databases using the basic local alignment search tool (BLAST), led to the identification of three novel MS4A members (MS4A13, MS4A14 and MS4A15) localised in the MS4A gene cluster on chromosome 11q12-13. MS4A13 encodes a putative transmembrane-four protein, while MS4A14 and MS4A15 were predicted to be transmembrane-three proteins with MS4A14 shown to have a highly divergent C-terminal domain that displayed significant similarity with a region of a Plasmodium falciparum heavy dynein chain. Comparative analysis of the translated amino acid sequences of these new MS4A members with CD20, Fc{u03B5}RI{u03B2}, HTm4 and MS4A5 showed high amino acid sequence identity between MS4A family members particularly within the transmembrane regions, with identity largely found in the first and second membrane-spanning domains. Further analysis suggested that many putative src-homology 2 (SH2) and 3 (SH3) binding domains and endosomal trafficking motifs were identified in MS4A family member amino acid sequences. Phylogenetic analysis showed that there is a clear distinction in sequence similarity between human MS4A members localised to chromosome 11q12-13 and the two distant relatives, TMEM176A and TMEM176B at chromosome 7q36.1. The mRNA distribution of the MS4A family in human tissues, primary cells (including the complete immune transcriptome) and transformed cell lines was investigated and indicated that MS4A genes are mostly found in the immune compartment and show differential expression within haematopoietic cell subsets. CD20, Fc{u03B5}RI{u03B2} and HTm4 showed haematopoietic cell expression distribution largely consistent with that previously described. MS4A4A, MS4A6A and MS4A7 were expressed across several subsets of haematopoietic cells, particularly in those that are involved in innate immunity including macrophages, neutrophils and dendritic cells. A subset of MS4A genes were found to be expressed in non-haematopoietic cells; MS4A5, MS4A13, MS4A14 and MS4A15 were all found in the testis and displayed differential distribution in cells that undergo spermatogenesis and those that are involved in homeostatic functions in the testis. Immunohistochemistry highlighted MS4A15 expression in the lung epithelium as well as cells of likely haematopoietic origins in the submucosal region of the lung. Within the airways, MS4A8B was identified predominantly at the apical surface of the tracheal and lung epithelium. MS4A8B was also found in cells of the subepithelium including cells with morphological similarities to mast cells. C-terminal tagging with enhanced green fluorescent protein (eGFP) and transient transfection into mammalian cell lines was undertaken for a number of MS4A members (MS4A4A, MS4A6A, MS4A7 and MS4A8B) in order to determine their subcellular localisation. MS4A4A and MS4A8B were expressed at the cell surface -a similar finding as previously reported for CD20, Fc{u03B5}RI{u03B2} and MS4A12. Intriguingly, MS4A6A and MS4A7 showed a distinctive pattern of localisation to that of other MS4A members, being identified near the cell surface in organelles juxtaposed to the plasma membrane. Further analysis of the specific subcellular localisation of MS4A4A, MS4A6A and MS4A7 suggested that they were differentially localised in the endocytic pathway. MS4A4A was localised to late and recycling endosomes (RE), MS4A6A appeared in clathrin coated pits and vesicles, early endosomes and RE and MS4A7 was present in RE. A truncated isoform of MS4A6A lacking a portion of the C-terminal domain was also identified and tagged with eGFP to determine its subcellular localisation. In contrast to the wild-type full-length form of MS4A6A, the truncated isoform was found to be expressed on the cell surface in a similar pattern as described for MS4A4A. Two putative tyrosine-based endosome sorting motifs were missing in the truncated isoform of MS4A6A, although mutation of the motifs in the wild-type form did not alter the subcellular localisation, suggesting these motifs were not responsible for the disparity in subcellular trafficking. The role of MS4A8B as a potential regulator of cell signalling was investigated. The stimulation of MS4A8B-transfected HMC-1 mast cells with IgE and antigen suggested that MS4A8B promoted the enhancement of tyrosine phosphorylation after high affinity IgE receptor engagement but inhibited degranulation. Similarly, the over expression of MS4A8B in BEAS2B bronchial epithelial cells was shown to enhance tyrosine phosphorylation after interleukin four treatment yet reduced the release of 15(S)-hydroxyeicosatetranoic acid (15(S)-HETE). In both of these systems, the mutation of the tyrosine residue in the C-terminal domain putative tyrosine-based signalling motif resulted in a reduction in tyrosine phosphorylation and enhanced inhibition of degranulation or 15(S)-HETE release, suggesting this is an activatory motif (ITAM). However, the results also suggest that MS4A8B is likely involved in inhibitory pathways. Finally, MS4A8B was studied as a promising potential candidate for the atopic asthma susceptibility gene localised at chromosome 11q13, due to its specific expression In the trachea and lung, in conjunction with the observed regulatory role in modifying Fc{u03B5}RI complex signalling in mast cells. Initial studies investigated whether there were any differences in the level or pattern of MS4A8B expression between lung biopsies from an asthmatic patient and a non-asthmatic control. No obvious expression changes were identified in the epithelium although expression of MS4A8B was not observed in the asthmatic subepithelium whereas it was noted in the non-asthmatic control. In conclusion, the data in this thesis adds to the information regarding the phylogeny, cellular and subcellular expression distribution and possible function for the MS4A family in signalling pathways. It also lays the foundation for the investigation of a possible novel atopy susceptibility gene at chromosome 11q13, in the form of MS4A8B.