Alfredo Ulloa-Aguirre

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

G protein-coupled receptors (GPCR) comprise the largest family of drug targets. This is not surprising as many signaling systems rely on this class of receptor to convert external and internal stimuli to intracellular responses. As is the case with other membrane proteins, GPCRs are subjected to a stringentquality control mechanism at the endoplasmic reticulum, which ensures that only correctly folded proteins enter the secretory pathway. Because of this quality control system, point mutations resulting in protein sequence variations may result in the production of misfolded and disease-causing proteins that are unable to reach their functional destinations in the cell. There is now a wealth of information demonstrating the functional rescue of misfolded mutant receptors by small nonpeptide molecules originally designed to serve as receptor antagonists; these small molecules (“pharmacoperones”) serve as molecular templates, promoting correct folding and allowing the mutants to pass the scrutiny of the cellular quality control system and be expressed at the cell surface membrane. Two of these systems are especially well characterized: the gonadotropin-releasing hormone and the vasopressin type 2 receptors, which play important roles in regulating reproduction and water homeostasis, respectively. Mutations in these receptors can lead to well defined diseases that are recognized as being caused by receptor misfolding that may potentially be amenable to treatment with pharmacoperones. This review is focused on protein misfolding and misrouting related to various disease states, with special emphasis on these two receptors, which have proved to be of value for development of drugs potentially useful in regulating GPCR trafficking in healthy and disease states.

Mutations of the 5α-reductase Type 2 gene in eight Mexican patients from six different pedigrees with 5α-reductase-2 deficiency

BACKGROUND AND OBJECTIVEu2003Male pseudohermaphroditism due to 5α‐reductase deficiency was originally described in 1974. Recently, 5α‐reductase Type 2 gene defects have been found generally to be due to point mutations within the 5 exons of the 5α‐reductase‐2 gene. In this report, we describe the molecular study of patients with 5α‐reductase deficiency.

Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: implications for the structure-function relationship of the FSH variants.

Follicle-stimulating hormone (FSH) is produced and secreted in multiple molecular forms. These isoforms differ in their oligosaccharide structures, which determine the particular behavior of a given variant in in vitro and in vivo systems. Employing heterologous cell assay systems, this and other laboratories have shown that highly sialylated human FSH variants exhibit lower receptor binding/immunoactivity as well as in vitro bioactivity/immunoactivity relationships than their less sialylated counterparts. It is not known, however, whether this characteristics behavior of the FSH isoforms is reproduced by homologous assay systems, in which unique variants of the receptor are presumptively expressed. To gain further insights into the structure–activity relationship of the various FSH isoforms, we analyzed the capacity of nine charge isoforms obtained after high-resolution chromatofocusing (pH window, 7.10 to <3.80) of anterior pituitary glycoprotein extracts to bind and activate their cognate receptor expressed by naturally occurring heterologous cell systems (rat granulosa cells and seminiferous tubule homogenates) as well as by human embryonic kidneyderived 293 (HEK-293) cells transfected with the human FSH (FSH-R) receptor cDNA. In both (heterologous and homologous) receptor assay systems, the isoforms displaced 125I-labeled FSH from the receptor in a dose-response manner; however, whereas in the heterologous systems, the receptor binding activity varied according to the elution pH value/sialic content of the isoforms, with the less acidic variants exhibiting higher receptor binding activity (r=0.851 and 0.495 [p<0.01 and p<0.05] for the granulosa cell and testicular homogenate receptor assay systems, respectively) than the more acidic/sialylated analogs, in the homologous assay, this relationship was practically absent (r=0.372, p N.S.). The capacity of the isoforms to induce androgen aromatization by rat granulosa cells followed the same trend shown by its corresponding receptor assay system (r=0.864, p<0.01). Interestingly and in contrast to the results observed in the homologous receptor binding assay, the ability of the isoforms to induce cAMP production by HEK-293 cells varied according to their elution pH value, with the more sialylated isoforms exhibiting lower potency than their less acidic counterparts (r=0.852, p<0.01). The results yielded by the heterologous assays suggest that the different potency of the isoforms to elicit abilogical effect in a naturally occurring receptor system depends primarily on the particular affinity of the receptor molecule for each isoform. The existence of a clear dissociation between receptor binding and signal transduction in the homologous system indicate that this later function is rather related to the different ability of the FSH glycosylation variants to induce and/or stabilize distinct receptor conformations that may permit preferential or different degrees of activation/ inhibition of a given signal transduction pathway. Thus, the human FSH receptor-transducer system apparently possesses sufficient versatility to respond in a different manner to glycosylation-dependent diverse FSH signals.

Evidence that human placenta is a site of sex hormone-binding globulin gene expression

The presence of an androgen-binding component in placenta was investigated in vitro using a tissue culture system of human placental explants. Explants of trophoblastic tissue from normal term placentas were kept in culture under appropriate conditions for at least 48 h in a serum-free medium. The existence of an androgen-binding protein was explored by binding assays, immunohistochemistry studies and Northern blot analyses of placental mRNA. Steady-state polyacrylamide gel electrophoresis and Scatchard plot analyses revealed the presence of a high affinity specific binding component for 5 alpha-dihydrotestosterone in cultured placenta. Immunohistochemical studies performed on intact placenta and on Percoll-gradient purified trophoblastic cells demonstrated the presence of specific immunoreactivity in the cytoplasm of syncytial cells. Northern blot analyses of placental mRNA showed a single hybridizable 32P-labeled human sex hormone-binding globulin (SHBG) cDNA band of approx. 1.6 kb which was identical in size to that obtained with liver mRNA. The results strongly suggest the placenta as an origin of SHBG and point out this tissue as an additional site of SHBG synthesis during pregnancy.

A novel homozygous mutation in the second transmembrane domain of the gonadotrophin releasing hormone receptor gene

BACKGROUND and OBJECTIVE Mutations in the GnRH receptor (GnRH‐R) gene cause hypogonadotrophic hypogonadism. Here, we present the molecular studies of the GnRH‐R gene in three families with isolated hypogonadotrophic hypogonadism.

A Naturally Occurring Basically Charged Human Follicle-Stimulating Hormone (FSH) Variant Inhibits FSH-Induced Androgen Aromatization and Tissue-Type Plasminogen Activator Enzyme Activity in vitro

It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED50 for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3′,5′-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.

No evidence of mutations in the follicle-stimulating hormone receptor gene in Mexican women with 46,XX pure gonadal dysgenesis

In the ovary FSH is necessary for normal follicular development, binding to its receptor (FSHR) that pertains to the superfamily of G-protein coupled receptors. In the FSHR gene, which consists of 10 exons, an homozygous mutation was reported in six Finnish families with gonadal dysgenesis; whereas two isolated French patients exhibited compound heterozygous mutations. Several groups, however, have searched for FSHR mutations, although in most cases the gene has been studied partially, not finding any genetic abnormalities in German, English, North American or Brazilian women. We performed direct sequencing of all 10 exons of the FSHR gene in seven sporadic patients and two sisters with 46,XX pure gonadal dysgenesis, to investigate the cause of their disorder. No heterozygous or homozygous mutant alleles were present in any of the patients. Although the number of patients evaluated was small, considering all the other previous reports, it seems that except in the Finnish population, the proportion of women with mutations in the encoding region of this gene is very low. Other possibilities for the presence of 46,XX gonadal dysgenesis, such as defects in the regulatory regions of the FSHR gene promoter, in the untranslated regions of exons 1 and 10, and within introns, or the existence of other genes likely to be important for normal ovarian function on the X chromosome or on autosomes, should be considered. In contrast with other studies, we did not find polymorphisms of the FSHR gene, indicating that apparently in Mexicans this gene is not highly polymorphic.

Contiguous gene syndrome due to deletion of the first three exons of the Kallmann gene and complete deletion of the steroid sulphatase gene

Large terminal or interstitial deletions of the 22.3 region on the short arm of the X chromosome cause contiguous gene syndromes. Kallmann syndrome (hypogonadotrophic hypogonadism with anosmia or hyposmia) associated with X‐linked ichthyosis, due to a contiguous gene syndrome, is an uncommon finding. Genetic defects have been demonstrated in the Xp22.3 region, explaining the presence of one or both entities in affected individuals. In this report we describe the molecular findings of a patient with Kallmann syndrome and X‐linked ichthyosis.

Normal testicular function without detectable follicle-stimulating hormone. A novel mutation in the follicle-stimulating hormone receptor gene leading to apparent constitutive activity and impaired agonist-induced desensitization and internalization.

Activating mutations in the follicle-stimulating hormone (FSH) receptor (FSHR) gene are rarely detected due to the absence of a clearly defined phenotype, particularly in men. We here report the biochemical features of a novel mutation in the first extracellular loop of the FSHR. The mutation (N431I) was detected in an asymptomatic man exhibiting normal spermatogenesis, suppressed serum FSH, and normal or elevated levels of biochemical markers of FSH action. Employing different experimental strategies on HEK-293 cells transiently expressing the N431I FSHR mutant, we found that the mutation led to decreased cell surface plasma membrane expression of the receptor protein, but conferred a low level of constitutive activity associated with markedly altered agonist-stimulated desensitization and internalization. These latter features may contribute and/or amplify the persistent activation of the receptor in both absence and presence of agonist and provide new insights into opportunities for adjuvant therapies based on disruption of these processes.

Evidence for an altered luteinizing hormone sensitivity to naloxone in pathological hyperprolactinaemia

OBJECTIVE The underlying mechanisms involved in the pathogenesis of amenorrhoea in hyperprolactlnaemic states still remain unclear. Conflicting information exists on the role of endogenous opiates on gonadotrophin disturbances in this pathological condition. In this study we have undertaken a detailed investigation of LH and PRL secretion before and during administration of naloxone, an opioid receptor blocker, in hyperprolactinaemic women with or without ovarian function In order to assess the role of ovarian steroids upon naloxone induced LH and PRL release.