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PubMed2001

minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel?

Inherited mutations and a polymorphism in minK-related peptide 1 (MiRP1) have been linked to congenital or acquired long-QT syndrome, pointing to the importance of MiRP1 in maintaining the cardiac electrical stability. We tested whether MiRP1 could affect the function of Kv4.x (x=2 and 3), the major pore-forming (alpha) subunits of transient outward (I(to)) channels in the heart. We used the Xenopus oocyte expression system to examine the effects of MiRP1 on Kv4.x channel gating kinetics and current amplitude and correlated these effects with MiRP1 expression level. MiRP1 slowed the rates of Kv4.2 activation and inactivation and shifted the voltage dependence of channel gating in the positive direction. These effects had a similar "dose" dependence: they plateaued at a cRNA ratio (MiRP1:Kv4.2) of 13:1, with half-maximum effects at estimated cRNA ratios of 2 to 4. On the other hand, MiRP1 had no significant effects on Kv4.2 current amplitude in the same range of expression level. When expressed at a comparable low level, MiRP1 had similar (although smaller) effects on Kv4.3 but could not modulate Kv1.4 (another alpha subunit of I(to) channels in the heart). Kv4.2 could be coimmunoprecipitated with epitope-tagged MiRP1, indicating that the 2 could form a stable complex. Our data suggest that MiRP1 may serve as a regulatory (beta) subunit of I(to) channels in the heart. This is supported by the observation that MiRP1 induced an "overshoot" of Kv4.2 current amplitude during channel recovery from inactivation, similar to the overshoot of I(to) described for human epicardial myocytes.

4-Aminopyridine↗
PubMed1996

Cell specific expression of human Bruton's agammaglobulinemia tyrosine kinase gene (Btk) is regulated by Sp1- and Spi-1/PU.1-family members.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase involved in the human disease X-linked agammaglobulinemia (XLA). The gene is expressed in all hematopoietic cells with the exception of T-cells and plasma cells. For this expression pattern the first 280 bp upstream of the major transcriptional start site seems to be sufficient. In vitro footprinting analysis within this part of the promoter revealed two Sp1 binding sites as well as a PU-box. The transcription factor Spi-1/PU.1 as well as the closely related factor Spi-B bound to the PU-box in B-cells. In the erythroleukemia cell line K562, due to the absence of Spi-B, only PU.1 bound to the Btk promoter. Mutation of either site reduced the expression in transient transfection experiments. However, mutation of the PU box had no effect in the T-cell line Jurkat, where none of the Spi-1 family members is expressed. In addition Spi-B as well as PU.1 were able to transactivate Btk expression. In fetal liver of PU.1-/- mice, which lack lymphoid and myeloid cells, expression of Btk was reduced two- to threefold but not abolished. Collectively this study shows that expression of the Btk gene is regulated by the combined action of Sp1- and PU.1-family members.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Transient restoration of gene rearrangement at multiple T cell receptor loci in gamma-irradiated scid mice.

The developmental arrest of thymocytes from scid mice, deficient in variable, (diversity), and joining, or V(D)J recombination, can be overcome by sublethal gamma-irradiation. Since previous studies focused on restoration of rearrangement of the T cell receptor (TCR) beta locus, productive rearrangement of which is selected for, we sought to examine to what extent locus specificity and cellular selection contributed to the observed effects. We report here that irradiation of newborn scid mice induces normal V-D-J rearrangements of the TCR delta locus, which like TCR beta, is also actively rearranged in CD(4-)CD(8-) (double negative) thymocytes. In contrast, no complete V-J alpha rearrangements were detected. Instead, we detected substantial levels of hairpin-terminated coding ends at the 5' end of the J alpha locus, demonstrating that TCR alpha rearrangements manifest the effects of the scid mutation. Irradiation, therefore, transiently compensates for the effects of the scid mutation in a locus-nonspecific manner in thymocytes, resulting in a burst of normal TCR beta and delta rearrangements. Irradiation also allows the development of cells that can initiate but fail to complete V(D)J recombination events at the TCR alpha locus, which is normally inaccessible in scid thymocytes.

Animals↗
PubMed1997

External cysteine residues in the serotonin transporter.

Hydropathy analysis predicts three cysteines (C109, C200, and C209) in extracellular loops of the rat serotonin transporter (SERT). We mutated these residues, singly and in combination, to either alanine or serine and expressed the mutant transporters in HeLa cells using the vaccinia-T7 transient expression system. Mutation of C109 to alanine had no effect on transport activity or surface expression of the transporter. In Na+-containing solutions, methanethiosulfonate (MTS) reagents had little effect on transport activity in the wild type or in the C109A mutant. When Na+ was replaced with Li+, inactivation of wild type by MTS reagents increased dramatically, but C109A was still resistant. The results suggest that C109 is exposed to the external medium in a manner dependent on cation binding. Replacing either C200 or C209 with serine resulted in either a partial (C200S) or almost total (C209S) loss of transport activity. MTS reagents rapidly inactivated transport activity in mutant C200S, suggesting increased accessibility of a previously unreactive cysteine residue. The double mutants C200S-C109A and C200S-C209S each retained partial activity. C200S-C109A was very sensitive to MTS reagents, but the C200S-C209S mutant was much less sensitive, similar to the wild type transporter. Replacement of C200 or C209 with serine dramatically decreased surface expression of the fully glycosylated transporter. Expression was normal, however, in the C200S-C209S double mutant. The Na+ dependence of transport and ligand binding was abnormal in both C200S and C200S-C209S mutants. Replacing C200 or C209 had similar effects on Na+ dependence and surface expression. Together with the increased MTS reactivity of C200S, these results support the possibility that C200 and C209 may be linked by a disulfide bond in the second external loop of SERT.

Animals↗
PubMed1995

Molecular cloning of mouse tissue inhibitor of metalloproteinases-3 and its promoter. Specific lack of expression in neoplastic JB6 cells may reflect altered gene methylation.

Mouse tissue inhibitor of metalloproteinases-3 (mTIMP-3), a gene specifically not expressed in neoplastic JB6 cells, have been isolated recently through the use of the mRNA differential display technique (Sun, Y., Hegamyer, G., and Colburn, N. H. (1994) Cancer Res. 54, 1139-1144). We report here the full-length mTIMP-3 cDNA sequence, the promoter sequence and partial characterization, expression and induction of TIMP-3, and the possible molecular basis for the lack of mTIMP-3 expression in neoplastic JB6 cells. There are three transcripts arising from alternative polyadenylation of mouse TIMP-3 gene, having sizes of 4.6, 2.8, and 2.3 kilobase pairs, respectively. All three TIMP-3 transcripts are expressed in preneoplastic but not neoplastic JB6 cells. Computer analysis of cloned TIMP-3 promoter revealed six AP-1 binding sites, two NF-KB sites, a c-Myc site, and two copies of a p53 binding motif separated by eight base pairs with two mismatches at the second motif, along with many other cis elements. TIMP-3 gene expression was inducible by AP-1 and NF-KB activators, 12-O-tetradecanoylphorbol-13-acetate, and tumor necrosis factor-alpha only in preneoplastic cells with an induction peak at 2 h post-treatment, suggesting classification of mTIMP-3 as a member of the immediate early gene family. Southern blot, mutational analysis, and transient transcriptional activation experiments revealed that the lack of expression of mTIMP-3 in neoplastic JB6 cells was due neither to gross deletion nor to promoter mutation of the gene, nor was there a lack of transcription factors required for transcriptional activation. Instead, the lack of TIMP-3 expression in neoplastic JB6 cells may reflect an abnormal methylation of the gene. Both hyper- and hypomethylation of the mTIMP-3 gene are associated with complete down-regulation of gene expression in neoplastic JB6 cell lines. Treatment of neoplastic cells with the methylase inhibitor 5-azacytidine caused reexpression of the mTIMP-3 gene in a tumor cell line that showed hypermethylation but not in another that showed hypomethylation of the gene, suggesting a complex role for methylation in the silencing of gene expression.

Animals↗
PubMed1999

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex.

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.

Alanine↗
PubMed1997

Regulation of the human TRH (hTRH) gene by human thyroid hormone receptor beta 1 (hTR beta 1) mutants.

TRH is negatively regulated by T3 both in the hypothalamic paraventricular nucleus and transient transfection models. Mutations in hTR beta 1 genes are associated with the syndrome of generalized resistance to thyroid hormone. To investigate potential effects of mutant TRs on T3 regulation of the hTRH gene, transient gene expression assays were performed in human neuroblastoma (HTB-11) cells with an hTRH promoter-luciferase construct, wild type (WT) hTR beta 1, and three qualitatively distinct hTR beta 1 mutant forms (ED, OK and PV). In the presence of T3 (10(-9) M), liganded WT-hTR beta 1 inhibited hTRH promoter activity significantly (40%). Cotransfection of each of the two mutants (ED and OK) achieved similar levels of inhibition only at 10 to 100 fold increased T3 concentrations. Of interest, a 10x excess of mutant ED or OK could also exert dominant negative effects upon WT hTR beta 1-T3 mediated inhibitory actions on the hTRH promoter. In contrast, mutant TR-PV exerted neither inhibitory nor dominant negative effects at even higher concentrations of T3. Moreover, all three unliganded mutant forms stimulated TRH promoter activity significantly in the absence of T3, despite their different mutations in the ligand-binding domain (LBD). These data demonstrate that thyroid hormone resistance at the level of TRH gene regulation, due to reduced inhibitory actions of mutant TR-T3 complexes, as well as dominant negative effects upon WT hTR beta 1 mediated inhibition, likely contribute to elevated TSH values observed in the syndrome of thyroid hormone resistance.

Dose-Response Relationship, Drug↗
PubMed2005

Inhibition of Kv4.3/KChIP2.2 channels by bupivacaine and its modulation by the pore mutation Kv4.3V401I.

BACKGROUND: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated. METHODS: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels. RESULTS: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 microm, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2. CONCLUSIONS: These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.

Anesthetics, Local↗
PubMed2005

GATA1 mutation and trisomy 21 are required only in haematopoietic cells for development of transient myeloproliferative disorder.

Trisomy 21 [Down's syndrome (DS)] and mutations in transcription factor GATA1 predispose neonates to a transient myeloproliferative disorder (TMD) and/or acute megakaryocytic leukaemia (AMKL). The role of trisomy 21 in their pathogenesis is unclear. We previously reported two rare neonates without DS who had TMD, one of whom progressed to AMKL. Trisomy 21 was detected only in blood cells at presentation with TMD/AMKL and disappeared with disease resolution. We now show that the blood cells at presentation of TMD harboured GATA1 genomic DNA mutations, suggesting a requirement for trisomy 21 in haematopoietic cells, rather than other cell types, for development of TMD/AMKL.

Base Sequence↗
PubMed2003

Two Italian kindreds with familial hypocalciuric hypercalcaemia caused by loss-of-function mutations in the calcium-sensing receptor (CaR) gene: functional characterization of a novel CaR missense mutation.

OBJECTIVES: Description of two unrelated Italian kindreds with familial hypocalciuric hypercalcaemia (FHH), an autosomal dominant disease mostly caused by heterozygous inactivating mutations of the Ca2+ sensing receptor (CaR). PATIENTS AND DESIGN: We studied 11 members of the two families. Genomic DNA was isolated from peripheral blood leucocytes in all family members and in 50 unrelated Italian controls. Total serum and ionized calcium, PTH, creatinine, phosphate, magnesium, and urinary calcium clearance to creatinine clearance ratio were measured. Direct sequencing of the entire coding region of the CaR was performed in the probands. Functional studies were performed in COS-7 cells transiently expressing the mutated CaR. RESULTS: In the proband of family A direct sequencing revealed a novel heterozygous Y218C missense mutation in exon 4. The same mutation was identified in the affected but not in the unaffected family members or in any of the 50 unrelated Italian controls. Transient expression of the Y218C CaR in COS-7 cells revealed a blunted Ca2+-evoked accumulation of inositol trisphosphates, indicating that the Y218C is a loss-of-function mutation. Cotransfection experiments showed that the mutant receptor had no impact on the function of the wild-type receptor, suggesting that a reduced expression of the normal CaR, rather than a dominant-negative effect, accounted for the functional impairment. In the proband of family B an already described heterozygous P55L missense mutation in exon 2 of the CaR gene was found. The same mutation was identified in the affected family members. CONCLUSIONS: We described two familial hypocalciuric hypercalcaemia kindreds with loss-of-function mutations of the Ca2+ receptor gene and identified a novel heterozygous mutation (Y218C) characterized by a blunted response to Ca2+ stimulation compared to the wild-type receptor and no interference with the function of the wild-type Ca2+ receptor.

Adult↗
PubMed1995

Adaptive mutation sequences reproduced by mismatch repair deficiency.

Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.

Bacterial Proteins↗
PubMed2006

Codon usage and selection on proteins.

Selection pressures on proteins are usually measured by comparing homologous nucleotide sequences (Zuckerkandl and Pauling 1965). Recently we introduced a novel method, termed volatility, to estimate selection pressures on proteins on the basis of their synonymous codon usage (Plotkin and Dushoff 2003; Plotkin et al. 2004). Here we provide a theoretical foundation for this approach. Under the Fisher-Wright model, we derive the expected frequencies of synonymous codons as a function of the strength of selection on amino acids, the mutation rate, and the effective population size. We analyze the conditions under which we can expect to draw inferences from biased codon usage, and we estimate the time scales required to establish and maintain such a signal. We find that synonymous codon usage can reliably distinguish between negative selection and neutrality only for organisms, such as some microbes, that experience large effective population sizes or periods of elevated mutation rates. The power of volatility to detect positive selection is also modest--requiring approximately 100 selected sites--but it depends less strongly on population size. We show that phenomena such as transient hyper-mutators can improve the power of volatility to detect selection, even when the neutral site heterozygosity is low. We also discuss several confounding factors, neglected by the Fisher-Wright model, that may limit the applicability of volatility in practice.

Algorithms↗
PubMed1992

Individual stage selector element mutations lead to reciprocal changes in beta- vs. epsilon-globin gene transcription: genetic confirmation of promoter competition during globin gene switching.

Biochemical and genetic analysis of the embryonic to adult beta-like globin gene switch in chickens has led to the hypothesis that competition between the promoters of the cis-linked epsilon- and beta-globin genes for interaction with a shared enhancer mediates the developmental changes in expression of beta-globin protein isotypes. To test specific predictions of this promoter competition model, a sensitive RNA/polymerase chain reaction assay has been used to investigate the effects of individual beta-globin promoter mutations on expression of the two linked genes in transiently transfected erythroid cells. Mutations that attenuated adult beta-globin transcription resulted concomitantly in a proportional increase in expression of the embryonic epsilon-globin gene. Consistent with the model, mutations disrupting the binding sites for either of two adult stage-specific transcription factors (NF-E4 and beta CTF) indicate that these sites are essential both for induction of beta-globin gene expression and for indirect suppression (through promoter competition) of epsilon-globin transcription in definitive (adult) erythroid cells. These results provide direct evidence that stage-specific transcription factors affect the equilibrium existing between multiple interacting globin cis-regulatory elements. We conclude that promoter competition is an important mechanism through which developmental regulation of chicken beta-globin gene switching is achieved and that such competitive interactions may prove to be generally applicable to the regulation of a variety of other temporally or spatially restricted gene expression patterns.

Animals↗
PubMed2005

Multiple endocrine neoplasia 2A due to a unique C609S RET mutation presents with pheochromocytoma and reduced penetrance of medullary thyroid carcinoma.

OBJECTIVE: We have identified a large kindred with multiple endocrine neoplasia 2A (MEN 2A) due to a mutation at RET codon 609 that results in a cysteine to serine substitution, a mutation previously identified in only one case in the literature. We characterized the clinical phenotype of the kindred and the biochemical mechanism of this new mutation. PATIENTS AND DESIGN: The index case, a 42-year-old woman, presented with pheochromocytoma. We screened 29 family members for the presence of the mutation. Of the 15 mutation-positive family members, 11 agreed to undergo further evaluation by physical examination, calcium and pentagastrin-stimulated calcitonin levels, measurement of urinary metanephrines, adrenal imaging and serum calcium levels. Biochemical characterization of the mutation was by transient transfection of human neuroblastoma cells and Western blot analysis. RESULTS: This kindred demonstrated an inheritance pattern consistent with autosomal dominant pheochromocytoma. Strikingly, no clinically evident case of medullary thyroid cancer (MTC) was observed among mutation-positive family members. Thyroidectomy in six cases revealed C-cell hyperplasia in all and microscopic MTC in two cases. Transfection experiments using human neuroblastoma cells showed that the mutant RET, unlike the wild-type receptor, is constitutively phosphorylated in the absence of ligand, and thus resembles other previously characterized MEN 2A mutations. CONCLUSIONS: The identification of a new mutation causing a MEN 2A phenotype that features pheochromocytoma and the surprising absence of clinically apparent MTC has significant implications for carriers of this mutation and provides further insights into the genotype-phenotype correlation in MEN 2A.

Adolescent↗
PubMed2002

Effects of Charcot-Marie-Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation.

Neurofilaments (NFs) are the major intermediate filaments (IFs) of mature neurons. They play important roles in the structure and function of axons. Recently, two mutations in the neurofilament light (NFL) subunit have been identified in families affected by Charcot-Marie-Tooth (CMT) neuropathy type 2. We have characterized the effects of these NFL mutations on the formation of IF networks using a transient transfection system. Both mutations disrupted the self-assembly of human NFL. The Q333P mutant in the rod domain of NFL also disrupted the formation of rat and human NFL/NFM heteropolymers. The phenotypes produced by the P8R mutation in the head domain of NFL were less severe. The P8R mutant NFL co-polymerized with NFM to form bundled filaments and, less often, aggregates. Our results suggest that alterations in the formation of a normal IF network in neurons elicited by these NFL mutations may contribute to the development of Charcot-Marie-Tooth neuropathy.

Amino Acid Sequence↗
PubMed2002

Homozygous mutation within the conserved Ala-Phe-Asn-Glu-Thr motif of exon 7 of the LH receptor causes male pseudohermaphroditism.

BACKGROUND: Human chorionic gonadotropin/luteinizing hormone (hCG/LH) function in the male is mediated by the LH receptor (LHR) and is crucial for the normal development of internal and external genitalia. We report a 46, XY patient who presented at the age of 16 with a female phenotype and delayed puberty. Gonads were located bilaterally in the inguinal canal, removed surgically and showed hypoplastic Leydig cells. Immunostaining for the LHR revealed that some Leydig cell progenitors were positive, while others were negative, reflecting different developmental stages of Leydig cell maturation. METHODS AND RESULTS: Molecular analysis of the LHR was performed on DNA extracted from blood samples of the patient, her parents and sister. The 11 exons of the LHR gene were amplified by PCR and subjected to further single stranded conformation polymorphism (SSCP) analysis. Aberrant migration patterns were observed in exon 7. Upon sequencing, a homozygous T to G transversion was identified, resulting in a F194V substitution located in the extracellular domain. The parents and sister were heterozygous carriers of this mutation. Functional studies in transiently transfected COS-7 cells with the F194V LHR mutation showed the lack of cAMP production upon hCG stimulation, indicating complete inactivation of the receptor due to impaired trafficking of the receptor to the membrane. The mutation is located within a stretch of five amino acids Ala (A)-Phe (F)-Asn (N)-Gly (G)-Thr (T), highly conserved in glycoprotein hormone receptors. For the follicle-stimulating hormone (FSH) receptor (FSHR) loss-of-function mutations have been allocated to this region, a homozygous A189V mutation resulting in a resistant ovary syndrome and impaired spermatogenesis and a heterozygous N191I mutation with no apparent phenotype. Further mutational and functional analysis of the AFN region in the LHR and FSHR revealed that the integrity of this amino acid sequence is crucial for receptor function.

Adolescent↗