Human Gonadotropin Alpha-subunit Research Articles

Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin alpha-subunit.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. The alpha-subunit has two N-glycosylation sites at Asn52 and Asn78. To obtain more information on the relationship between the structure and function of the alpha-subunit, we introduced a novel N-glycosylation site in the N-terminal region by mutating Asp3 and Gln5 into Asn and Thr, respectively. Glycosylation mutants were expressed alone or with hCGbeta-subunit in Chinese hamster ovary cells. New N-linked oligosaccharides were efficiently added to the wild-type and mutant alpha-subunits lacking N-glycan at Asn52 (alpha deltaAsn1), Asn78 (alpha deltaAsn2), and both (alpha deltaAsn(1 + 2)). The new sugar chain did not affect secretion and assembly except that 1) it increased the intracellular degradation of alpha deltaAsn(1 + 2), and 2) it augmented the assembly of alpha deltaAsn1 with hCGbeta-subunit. Amino acid changes generated the attachment of O-glycosylation in free alpha-subunit but not in assembled form. These data indicate that the newly introduced N-glycosylation consensus sequence is functional, and that the N-terminal region of the alpha-subunit is flexible and can be modified without affecting the intracellular function. Furthermore, amino acid sequences in the N-terminus are involved in the O-glycosylation in free alpha-subunit.

Transcriptional activation of the gonadotropin-releasing hormone receptor gene by activin A.

It has been reported that activin A stimulates the synthesis of the GnRH receptors (GnRHR) in rat pituitary cultures. However, the role of activin A in the regulation of the GnRHR gene at the molecular level is not known. In the present work, we have studied the regulation of the GnRHR gene by activin A in the gonadotrope cell line, alpha T3-1, where the GnRHR gene is highly expressed. First, we demonstrate that these cells express the mRNAs of three types of activin receptors: I, II, and IIB. Activin A increases GnRHR mRNA levels in a dose-and time-dependent manner, with maximal stimulation (2.5 +/- 0.5-fold) occurring with a dose of 20 ng/ml after 36 h of incubation. To ascertain whether this effect occurs at the transcriptional level, we performed nuclear run-off experiments in alpha T3-1 cells, which demonstrate a 1.6-fold increase in the levels of newly synthesized GnRHR mRNA in response to activin A. To investigate further the effect of activin A on the transcription of the GnRHR gene, alpha T3-1 cells were transiently transfected with a mouse GnRHR promoter/luciferase reporter gene (GnRHR-Luc) and challenged with activin A. Luciferase activity increases in response to activin A to the same extent (2.4 +/- 0.4-fold) and with similar dose-response and time-course profiles as the mRNA levels. Follistatin (100 ng/ml), a well known activin antagonist, completely abolishes the activin A effect on both mRNA levels and GnRHR-Luc activity. Follistatin also decreases the basal expression of the GnRHR gene by 33% as determined by GnRHR-Luc activity. This, together with our demonstration of the presence of the inhibin beta B-subunit mRNA in alpha T3-1 cells, suggests a potential paracrine/autocrine role of endogenous activin B on the regulation of the GnRHR gene in these cells. To provide evidence for biological significance of activin A stimulation of GnRHR gene expression, the response of a human gonadotropin alpha-subunit promoter/luciferase reporter gene (alpha Gon-Luc) to GnRH was assessed in alpha T3-1 cells pretreated with activin A. Activin enhances the stimulation of alpha Gon-Luc activity by GnRH by 1.6 +/- 0.4-fold. These data demonstrate that activin A can stimulate the expression of the GnRHR gene at the transcriptional level. Furthermore, transfection studies localize the activin responsive element to 1.2 kb of the 5'-flanking region of the GnRHR gene. Transcriptional activation of the GnRHR gene by activin A may serve as a mechanism for the modulation of gonadotrope responsiveness to GnRH.

Role of the Pro-Leu-Arg motif in glycosylation of human gonadotropin alpha-subunit.

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunit. Processing of the N-linked oligosaccharide of the glycoprotein family is both tissue and dimer specific. LH, TSH, and free alpha synthesized in pituitary bear oligosaccharide terminating with sulfate (SO4) and N-acetylgalactosamine (GalNAc), whereas the termination of oligosaccharide in CG synthesized in placenta and FSH is sialic acid and galactose (Gal). Using site-directed mutagenesis and gene transfer, we studied the role of the Pro-Leu-Arg motif, which has been shown to be a recognition marker of glycoprotein hormone-specific GalNAc transferase, in sulfation of N-linked oligosaccharide in alpha-subunit. The wild-type or mutated alpha gene was transfected into GH3 cells. Our data revealed that substitution of the Pro-Leu-Arg motif by Ala-Leu-Ala did not affect the sulfation of N-linked oligosaccharide, but generated the attachment of O-linked oligosaccharide. alpha-Subunit containing either of the two N-linked glycosylations is also sulfated. We conclude that in GH3 cells, the Pro-Leu-Arg motif plays no role in the sulfation of oligosaccharide in alpha-subunit, and both N-glycosylations are terminated with SO4.

Role of the Pro-Leu-Arg motif in glycosylation of human gonadotropin alpha-subunit

Role of the Pro-Leu-Arg motif in glycosylation of human gonadotropin alpha-subunit

Mutagenesis of cysteine residues in the human gonadotropin alpha subunit. Roles of individual disulfide bonds in secretion, assembly, and biologic activity.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha subunit but differ in their hormone-specific beta subunits. Site-directed mutagenesis was used to examine the role of the five disulfide bonds in the alpha subunit on the folding, assembly with the hCG beta subunit, and in cases where dimer formation occurred, receptor binding and signal transduction. Cysteine residues in the disulfide bonds formed by cysteines 7-31, 10-60, 28-82, 59-87, and 32-84 (Lapthorn, A., Harris, D. Littlejohn, A., Lustbader, J. Canfield, R., Machin, K., Morgan, F., and Isaacs, N. (1994) Nature 369, 455-461) were converted to alanine, and these mutants were transfected alone or together with the wild-type hCG beta gene into Chinese hamster ovary cells. The alpha Cys-10, 28, 60, 82, and 84 mutants were not secreted and in most cases were degraded at a faster rate than the native subunit. In addition, these mutants failed to assemble with the hCG beta subunit. Mutants with alterations at alpha Cys-7, 31, 32, 59, or 87 were secreted and combined with the beta subunit. Heterodimers containing a 7-31 double mutant bound to human lutropin-chorionic gonadotropin receptor expressed in transfected human fetal kidney cells, and stimulated cAMP comparable to wild-type hCG. Dimers containing the beta subunit with either single mutant alpha 59, alpha 87, alpha 32, or the alpha 59-87 double mutant showed much lower affinity for the receptor than wild-type hCG. These results suggest that disulfide bonds associated with alpha 7, alpha 31, alpha 59, alpha 87, and alpha 32 are not essential for the alpha subunit to fold into a form that will combine with the hCG beta subunit and to produce a biologically active dimer. This contrasts with observations of the hCG beta subunit where all the disulfide bonds are required for efficient combination and folding (Suganuma, N., Matzuk, M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307). In addition, the lack of secretion of some mutants reflects previous observations that proteins which do not fold correctly are rapidly degraded. Thus, alpha subunit mutants which fold properly are secreted and can form heterodimers.

GATA-binding proteins regulate the human gonadotropin alpha-subunit gene in the placenta and pituitary gland.

The human glycoprotein hormone alpha-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some of which are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases alpha-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in alpha T3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the alpha-subunit gene GATA element. In alpha T3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into alpha T3-1 cells activates the alpha-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary alpha-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.

Expression and cAMP-mediated regulation of the human gonadotropin alpha subunit gene in transfected mouse L-cells

Abstract Expression of the dimeric glycoprotein hormone, human chorionic gonadotropin (HCG), occurs either eutopically in placental trophoblast cells and trophoblastic tumor cells (choriocarcinoma) or ectopically in nontrophoblastic tumor cells. However, regulation of constitutive HCG-subunit mRNA production appears to differ in trophoblastic and nontrophoblastic cells, as evidenced by the fact that cAMP analogs and agonists enhance cutopic but not ectopic HCG-subunit mRNA synthesis. In the present study, we compared the effects of cAMP on HCG α-subunit expression in human choriocarcinoma cells and in nontrophoblastic mouse L-cells stably transfected with the HCG α-subunit gene. Constitutive levels of α-subunit expression in transfected mouse L-cells were equivalent to or exceeded those found in choriocarcinoma cells as determined by Northern blot analysis and indirect immunofluorescence for α-subunit protein. However, cAMP-mediated induction of α-subunit gene expression was retained in nontrophoblastic L-cells and closely paralleled that observed in human choriocarcinoma cells. These findings indicate that cells distinctly nontrophoblastic in origin may share the necessary cellular factors for cAMP-mediated induction of α-subunit gene expression. Failure of ectopic HCG-producing tumor cells to be stimulated by cAMP may thus be the result of deletion or mutation of such factors.