Molecular Human Reproduction, Vol. 8, No. 3, 201-212,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
A novel Ala–3Thr mutation in the signal peptide of human luteinizing hormone ß-subunit: potentiation of the inositol phosphate signalling pathway and attenuation of the adenylate cyclase pathway by recombinant variant hormone
1 Department of Physiology, Institute of Biomedicine and 2 Department of Biotechnology, University of Turku, 20520 Turku, Finland and 3 Department of Biological Sciences, Florida International University, Miami, FL 33199, USA
Abstract
Upon screening for polymorphisms in the human luteinizing hormone ß-subunit (LHß) gene, we discovered a novel mutation in the LHß signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G52A point mutation in exon 2 of the LHß gene, detected in heterozygous form in several normal DNA samples, caused an Ala–3Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala–3Thr variant (V) LH were produced in human embryonic kidney (HEK) 293 cells and purified. The immunoreactivities of the recombinant LH were determined by immunofluorometric assays and in-vitro bioactivities in mLTC-1 cells were assessed by using cAMP, progesterone and inositol trisphosphate (IP3), and activation of mitogen-activated protein kinase (MAPK) as end-points. Whereas both LH forms stimulated progesterone production and MAPK in similar fashion, WT-LH was more potent in stimulating cAMP, and V-LH was more potent in stimulating IP3 generation. Both LH forms bound to LH receptors with similar affinities. No evidence was found for influence of the signal peptide mutation on efficacy of 
- and ß-subunit dimerization. Sequencing of the recombinant V-LHß protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala–3Thr mutation in the LHß-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.
luteinizing hormone/LHß variant/mutation/PCR/purification
Notes
4 To whom correspondence should be addressed at: Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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