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Constitutive Activity in Receptors and Other Proteins, Part A, Volume 484
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Constitutive Activity in Receptors and Other Proteins, Part B, Volume 485
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Flexible - Read on multiple operating systems and devices. Easily read eBooks on smart phones, computers, or any eBook readers, including Kindle. We, therefore, tested whether this nucleotide variation, which generates a recognition site for the restriction enzyme Mnl I, would represent a frequent polymorphism. The sequence variation, present in the homozygous state, is a C-to-A transversion located within the first GHSR exon c.
C Conservation of the GHSR1a amino acid sequence among species within the region bracketing the mutation site between the fourth TM4 and the fifth TM5 transmembrane domains; AE is shown by an arrow. This GHSR nucleotide substitution p. AE predicts the replacement of an apolar and neutral residue alanine by a polar and charged amino acid glutamate.
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At the protein level, this missense mutation is located in the second extracellular loop of the GHSR1a protein Figure 1 B and involves a residue totally conserved in all species studied so far Figure 1 C. Such a high degree of conservation of alanine , therefore, suggests that amino acid substitutions at this position could be detrimental to receptor function. The segregation of this sequence variation with the short stature phenotype was subsequently analyzed within the 2 families.
DNA samples of available individuals were amplified with the use of primers bracketing the mutation site, and the resulting PCR products were then digested by Mnl I Figure 2. In family 1 Figure 2 A , in which the proband with ISS individual II2 was found to carry the AE mutation in the homozygous state, both parents individuals I1 and I2 are heterozygous for this mutation, a result in keeping with the consanguinity documented in that family; this analysis revealed that all 3 siblings individuals II1, II3, and II4 were also heterozygous for the mutation.
Both parents have a significant height reduction: the father and the mother reached a final height of cm and cm 3. In family 2 Figure 2 B , in which the proband with IGHD individual II1 was found to carry the same mutation in the heterozygous state, the father, who has a slight height reduction 2.
A Family 1. B Family 2. Circles and squares denote female and male family members, respectively. The SD to mean height for age is given below each symbol; height values are before GH treatment. Black symbols denote a short stature.
The probands are indicated by arrows. Details of the clinical and biological phenotypes of the children with short stature from these 2 families are presented in Table 2 , according to genotype.
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Serum levels of IGF-1 were found to be low in the patient from family 2 individual II1 , whereas, in spite of the documented growth delay, they were found to be within the normal range in all 3 patients from family 1 individuals II2, II3, and II4. Finally, among the children with short stature, the 3 who received a GH treatment increased their growth velocity data not shown. The growth curve of the homozygous patient family 1-II2 shows that, although her length was normal at birth 50 cm , a growth delay appeared within the first 2 years of life and worsened with age until the beginning of GH therapy 3.
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As for her weight, it remained within 2 SD below the normal mean for age until the age of 9 years and then increased continuously so that her BMI reached the overweight limit for age after the age of 13 years Figure 3 and Table 2. Her parents, who carry the AE mutation in the heterozygous state, are obese, whereas her heterozygous siblings display either a slight overweight II1 or a trend to overweight II3 and II4 Table 2 and data not shown.
In contrast, heterozygous individuals from family 2 have a BMI within the normal range, the proband II1 even being rather lean. Height cm and weight kg curves of the proband family 1, patient II2. The GH treatment is shown by the gray area. Clinical and biological phenotypes of the children with short stature from families 1 and 2 before GH treatment, according to genotype. Finally, to assess the affinity of GHSR1a AE for I-ghrelin — the low levels of binding to this mutant receptor in transient transfection experiments precluding any affinity measurements — we generated HEK cell lines stably expressing either the WT or the AE receptor.
The specific binding, which represents the difference between total binding and nonspecific binding, is expressed as a percentage of the binding associated with the WT GHSR1a receptor.
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A representative experiment of 3 independent experiments each performed in triplicate is shown. The binding of 30 pM of I-ghrelin was displaced by increasing concentrations of cold ghrelin. The immunostaining of HA-tagged GHSR1a receptors was performed by means of an anti-HA monoclonal primary antibody incubated in the absence or in the presence of a permeabilizing reagent in order to visualize the receptors located at the cell surface or at both the cell surface and the intracellular level, respectively.
These experiments revealed a peripheral plasma membrane staining on nonpermeabilized cells expressing the WT or the mutant receptor, and after plasma membrane permeabilization, both transfectants exhibited intense cytoplasmic staining. Consistent with this hypothesis, the number of stained cells examined after transfection of HA-GHSR-AE and plasma membrane permeabilization was slightly higher than that of cells expressing the WT plasmid.
Overall, these observations support the fact that the AE mutant is efficiently translated into a protein, but with only a small fraction properly expressed at the cell surface, this plasma membrane fraction displaying a normal affinity for ghrelin. To test whether the AE mutant GHSR1a displays the same property, given its decreased cell-surface expression, we monitored the constitutive activity of the WT and the mutant GHSR1a as a function of their own cell-surface expression.
Both the WT and the mutant receptors were tagged at their N-terminus with an HA epitope, in order to assess their basal activity relative to their cell-surface expression. This experiment, shown in Figure 5 A, disclosed the following features. In contrast, a similar experiment performed with the AE receptor showed a much lower increase in basal activity Figure 5 A. Taken together, these data, which reflect the degree of ligand-independent signaling activity of the GHSR1a, therefore, demonstrate that the AE mutation leads to a loss of constitutive activity of the receptor, which results from both its intrinsic inability to signal constitutively and its decreased cell-surface expression.
Signals associated with cells transfected with the mock plasmid have been subtracted, so that plotted signals represent specific RLU. To investigate the molecular mechanism underlying the dominant mode of inheritance of the short stature phenotype, we assayed transcription from the same POU1F1 -Luc reporter gene in the presence of both WT and mutant GHSR proteins. As shown in Figure 5 B, cotransfection of equal amounts of the plasmids encoding these 2 receptors did not inhibit the constitutive activity associated with the WT receptor, thereby suggesting the absence of a dominant-negative effect of the mutant AE over the WT GHSR1a, at least in that system.
To further characterize the biological properties of this particular mutant, we assessed its ability to activate the CRE pathway in response to different ligands. The results confirmed that the basal activity of the mutant receptor — assayed in the absence of any ligand — was undetectable Figure 6 A. Indeed, as shown in Figure 6 A, this molecule did not change the basal activity of the mutant receptor, which remained within background levels.
A CRE-mediated transcriptional activity. Cells were cotransfected with ng of each expression plasmid and ng of the pPOU1F1-Luc reporter plasmid.
limamamacley.gq B SRE-mediated transcriptional activity.