Background: 20% of the familial isolated pituitary adenoma (FIPA) population harbour an aryl hydrocarbon receptor-interacting protein (AIP) gene mutation. The recognition of whether a variant is pathogenic can be difficult, in cases where the observed change does not lead to a truncated protein. Segregation with disease in a family, in silico predictions, loss of heterozigosity in the tumour, in vitro functional studies and screening of the variant in controls may help in ascertaining the pathogenicity, but results can be missing or inconclusive.
Aim: To study the half-life of wild type and variant AIP proteins in transfected GH3 cells.
Methods: GH3 cells were transfected with either myc- or flag-tagged wild-type or mutated AIP constructs. After 24 hours, transfected GH3 cells were treated with translation inhibitor cycloheximide. Proteins were extracted at 1, 2, 5, 9, 12, 24, ad 30 hours and immunoblotted for the tagged protein and houskeeping gene GAPDH.
Results: Wild-type AIP has an in vitro half life of 20.9 hours, while the half life of the truncation mutation R304X was only 1.2 hours in our experimental system. AIP R16 h and R304Q behaved similarly to wild-type AIP protein while A299V (T1/2=3.7 h) and I257V (T1/2=6.3 h) had a significantly reduced half life.
Conclusion: The half-life of some variant AIP proteins was significantly reduced compared to wild-type protein, suggesting significant alterations in the protein stability in vitro. We hypothesise that some full-length AIP variants may predispose to pituitary adenoma development due to a shortened half-life.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.