Abstract
We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.
Highlights
Interleukin (IL)1-15 is a cytokine that was discovered through its capacity to replace IL-2 in supporting the growth of the murine IL-2-dependent CTLL cell line [1, 2]
IL-15 stimulates the proliferation of murine mast cell lines, but this effect is mediated through a novel, as yet unidentified, receptor not involving IL-15R␣ or IL-2 receptor (IL-2R) and IL-2R␥ [20]
Using oligonucleotide primers corresponding to the N-terminal end of exon 1 and the C-terminal end of exon 7 or 7Ј, RTPCR amplifications were carried out on mRNAs prepared from different cell lines and tissues (Fig. 2)
Summary
Total RNA was extracted from various human cell lines and tissues using guanidinium thiocyanate/phenol as described [21]. Total RNAs from human liver, brain, and small intestine were purchased from CLONTECH (Basingstoke, United Kingdom). Reverse transcription and PCR amplifications were performed as described previously [22]. For PCR, the cycling conditions were as follows: denaturation for 1 min at 94 °C; annealing for 1 min at 66 °C (E1/E7), 68 °C (E1/E7Ј), 52 °C (E4/p3BGH), 55 °C (5Ј/3Ј), or 60 °C (5E4.1/E7.2); and elongation for 1 min at 72 °C (30 cycles). The following primers were used: E1, 5Ј-AGTCCAGCGGTGTCCTGTGG; E7, 5Ј-TCATAGGTGGTGAGAGCAGT; E7Ј, 5Ј-TCAACAGACGCTTCCCACTG; E4, 5Ј-GAACTCACAGCATCCGCC; p3BGH, 5Ј-TAGAAGGCACAGTCGAGG; 5Ј (sense), 5Ј-CGTGCTGCTGACCGAGGCC; 3Ј (antisense), 5Ј-TTCGTGGATGCCACAGGAC; 5E4.1, 5Ј-GCAGCTTCATCTCCCAG; and E7.2, 5Ј-TAGGTGGTGAGAGC
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