Personnel
Back Row: Heather Powell, Kelli Black, D. Randy McMillan
Front Row: Daniela Rogoff, Michele Hutchison, Perrin White
Projects
Biochemical Basis of Cortisone Reductase Deficiency
Studies of Very Large G-protein Coupled Receptor-1 (VLGR1)
Personnel
Perrin C. White, M.D.—Principal Investigator
Bibliography
Dr. White has studied the molecular basis of several genetic diseases of steroid hormone synthesis and metabolism over the years. Some of these diseases affect growth, sexual differention or the ability to conserve salt in the blood, whereas others cause high blood pressure. Most recently, he has branched into study of a very large cell-surface molecule that his laboratory discovered serendipitously. It is important for development of the nervous system; mutations in this protein cause seizures, as well as, deafness and progressive blindness (Usher syndrome).
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D. Randy McMillian, Ph.D.—Assistant Professor
Bibliography
Dr. McMillan's interests involve the role of G protein-coupled receptors (GPCR) in development. He and Dr. White have cloned the gene that codes for the largest known GPCR, the Very Large G protein coupled Receptor 1 (VLGR1). Presently, he is involved in characterizing the function of VLGR1 to determine its role in development of the mammalian central nervous system.
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Daniela C. Rogoff, M.D., Ph.D.—Post Doctoral Researcher
Bibliography
Dr. Rogoff was trained in Buenos Aires, Argentina. During her residency she became interested in adrenal disorders and focused her research on this subject. She began working in Dr. White's laboratory in September 2005. Her current project involves the study of the effect of specific cofactors in glucocorticoid action.
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Technicians
Kelli A. Black (Research Associate)
Bibliography
Heather M. Powell (Research Technician)
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Projects
Biochemical Basis of Cortisone Reductase Deficiency
Apparent cortisone reductase deficiency (ACRD) is a human disease in which the metabolic clearance of cortisol is increased, leading to overactivity of the adrenal cortex, increased secretion of adrenal androgens, and a phenotype similar to polycystic ovary syndrome (PCOS)/metabolic syndrome/syndrome X. Our previous work has shown that ACRD is a digenic disease, requiring carriage of mutations in two genes, HSD11B1 (encoding the type 1 or liver isozyme of 11β-hydroxysteroid dehydrogenase) and H6PD (encoding hexose-6-phosphate dehydrogenase). We propose to elucidate the mechanisms by which these mutations interact to cause disease. We will define the expression of H6PD in a variety of murine and human tissues by measuring mRNA levels using quantitative PCR, and by measuring protein expression using immunoblotting and assays of enzymatic activity. We will investigate the direct role of H6PD in determining the set point of 11β-HSD1 activity by expressing H6PD and 11β-HSD1 in bacteria, and by analyzing the effect of varying H6PDH expression upon 11β-HSD1 oxo-reductase activity in mammalian cells. We will determine functional consequences of variations in H6PD activity upon phenotype of primary human adipocytes and hepatocytes. We will develop a mouse model of ACRD. To do this, we will produce a mouse with a targeted inactivation of H6PD, and cross this to a mouse with an inactivated HSD11B1 allele. We will breed double heterozygous mutant mice to produce a mouse model of ACRD. Phenotypic characterization will focus on gene and protein expression, 11β-HSD1 activity and equilibrium set-point in liver and adipose tissue, development of the adrenal cortex and levels of expression for key genes regulating steroidogenesis, assays of adrenal function, adipose tissue development, and hepatic enzyme expression. Finally, we will genotype women with polycystic ovary syndrome to determine the degree to which polymorphisms in the human HSD11B1 and H6PDH genes are risk factors for the development of this condition. Subsets of the genotyping data will be analyzed as an association study, as an affected sib pair study, and by transmission disequilibrium testing. Additional polymorphisms in these genes will be sought in PCOS subjects.
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Studies of the Very Large G-protein Coupled Receptor-1 (VLGR1)
Structure
Expression and Functions
A Role in Hearing
A Structural Mechanism
Functional Consequences of VLGR1 Mutations
Structure
The orphan 7-transmembrane segment receptor, VLGR1, is the largest known cell-surface protein. VLGR1 is a member of the secretin family (family 2 or B) of G-protein coupled receptors and is distinguished by a very large ectodomain consisting mainly of 35 calcium-binding Calx-β repeats that resemble the regulatory domains of sodium-calcium exchangers. VLGR1 is presumably anchored to the plasma membrane by its seven transmembrane domains and cytoplasmic tail.
If the Calx-β repeats were in an extended conformation, the ectodomain would project ~180nm from the surface of the cell. The C-terminal residues contain a consensus motif (Ser/Thr)-Xaa-(Val/Ile/Leu) recognized by PDZ domain proteins, which act as scaffolds for assembling signal transduction proteins into functional signaling units. VLGR1 is highly conserved across evolution and we have cloned the ~19.0Kbp cDNA from human, mouse and zebrafish.
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Expression and Function
VLGR1 is strongly expressed during development, with the highest levels seen in the fetal ventricular zone and retina. Temporal expression during neurogenesis suggests VLGR1 may function in regulating development of the vertebrate central nervous system. Naturally occurring mutations have been found in VLGR1, which cause audiogenic (sound generated) seizures in mice and Usher syndrome Type 2C in humans. Usher syndrome consists of sensorineural deafness, retinitis pigmentosa and, in some forms, vestibular dysfunction. It has a frequency of 1/25,000 in the United States and includes three subtypes numbered in descending order of severity with type 2 being the most frequent.
To determine the function of VLGR1, we have generated a mutant mouse (VLGR/del7TM) in which the transmembrane and cytoplasmic domains of the receptor have been removed. The resulting mutant mice are susceptible to audiogenic seizures, produced by intense sound insult, until approximately 6 weeks of age and then become resistant. Auditory brainstem response assays indicate that the mutant mice are profoundly hearing impaired by P20, the earliest stage tested, and acoustic sensitivity continues to decrease over the following month, resulting in deafness.
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A Role in Hearing
The sensory hair bundle in the inner ear is a mechanosensitive structure located at the apical pole of the sensory hair cell. The hair bundle is composed of numerous modified microvilli (stereocilia) that are arrayed in rows of increasing height across the apical surface of the hair cell. Stereocilia are coupled to one another by a number of different link types. Four morphologically and biochemically distinct link types can be distinguished extending between the stereocilia of hair bundles: tip links, horizontal top connectors, shaft connectors and ankle links. Tip links are thought to gate the hair cell’s mechanotransducer channel,whereas other link types may serve to maintain the structural integrity of the hair bundle and/or orchestrate its development.
The ankle link antigen is an epitope specifically associated with ankle links, at the base of the stereocilia and the calycal processes of retinal photoreceptors. We have conclusively identified the ankle link antigen as VLGR1.
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A Structural Mechanism
Ankle links fail to form in the cochleae of VLGR1/del7TM mice and the bundles become disorganised just after birth, ultimately resulting in deafness. In homozygous mutant mice a phenotype first becomes readily apparent at P2, when fully-formed ankle links are first observed by transmission electron microscopy and ~7-8 days after the hair bundle has first emerged. VLGR1 is therefore not required for the initial stages of hair bundle genesis, but appears to be critically involved in maintaining the form of the bundle once it has reached a certain stage of development. In its absence, cochlear hair bundles lose their tightly defined V-shaped configuration and become poorly aligned. Surprisingly, although developing vestibular hair bundles have ankle links and express VLGR1, these cells do not appear to be dependent upon VLGR1 for their development and long-term survival.
The molecular interactions by which VLGR1 ectodomains form ankle links remain to be defined. Although individual filaments of the ankle link complex appear to be single stranded, the particles observed within the central density of the ankle link complex could represent points where ectodomain modules interact. VLGR1 could interact in trans, homophilically, mediated by the Calx-β repeats.
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Functional Consequences of VLGR1 Mutations
The physiological phenotype of the VLGR1/del7TM mutant mice is similar to that observed in humans diagnosed with Usher syndrome 2C. The pathology is also progressive in the VLGR1/del7TM mouse, with early hair bundle defects leading to a complete loss of hair cells in the basal turn of the cochlea by 2 months of age. The normal gait and balance behavior of VLGR1/del7TM mutant mice, and the normal transduction currents measured in their vestibular hair cells, also correspond to the lack of vestibular dysfunction seen in humans with Usher syndrome 2C.
Future studies will be directed at understanding how the different expression patterns are determined and how the individual proteins control the various phases of hair-bundle morphogenesis.
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