A novel Ala(-3)Thr mutation in the signal peptide of human luteinizing hormone beta-subunit: potentiation of the inositol phosphate signalling pathway and attenuation of the adenylate cyclase pathway by recombinant variant hormone.
Upon screening for polymorphisms in the human luteinizing hormone beta-subunit (LH beta) gene, we discovered a novel mutation in the LH beta signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G(52)A point mutation in exon 2 of the LH beta gene, detected in heterozygous form in several normal DNA samples, caused an Ala(-3)Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala(-3)Thr 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 (IP(3)), 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 IP(3) 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 alpha- and beta-subunit dimerization. Sequencing of the recombinant V-LH beta protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala(-3)Thr mutation in the LH beta-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.
Jiang
Min
M
Department of Physiology, Institute of Biomedicine, University of Turku, 20520 Turku, Finland.
Lamminen
Tarja
T
Pakarinen
Pirjo
P
Hellman
Jukka
J
Manna
Pulak
P
Herrera
Rene J
RJ
Huhtaniemi
Ilpo
I
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011110:Polymorphism, Genetic; D011374:Progesterone / Q000096:biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics; D011974:Receptors, LH / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003470:Culture Media; D015292:Glycoprotein Hormones, alpha Subunit; D007295:Inositol Phosphates; D021382:Protein Sorting Signals; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D013912:Threonine; D011374:Progesterone; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D000409:Alanine