BAI2 gehört zu den Adhesion-G-Protein-gekoppelten Rezeptoren (aGPCR). Diese bisher wenig untersuchte Klasse von ca. 30 GPCR ist charakterisiert durch eine komplexe genomische Struktur, sehr große extrazelluläre Domänen und eine Vielzahl von Splicevarianten. Bisher ist bei den meisten aGPCR, wie auch bei BAI2, wenig über ihre Signaltransduktion und Funktion bekannt. Zum Verständnis der physiologischen Relevanz und zur Suche nach dem endogenen Agonist sind Kenntnisse über Proteinstruktur, Splicevarianten und Signaltransduktion essentiell. Ziel dieser Arbeit war es, mittels verschiedener in vitro-Methoden die Proteinstruktur des BAI2 in den transmembranären und intrazellulären Domänen näher zu untersuchen, sowie die natürlichen Splicevarianten in diesem Bereich, deren evolutionäre Konservierung, Gewebespezifität und Quantität zu erfassen. Für beide gefundenen Splicevarianten, eine im dritten intrazellulären Loop (ICL3) und eine im C-Terminus, konnte eine evolutionäre Konservierung auf Aminosäure- und genomischer Organisationsebene, sowie ihre Entstehung durch Exonskipping nachgewiesen werden. Nachfolgend wurden die Splicevarianten auf mögliche Interaktionen mit intrazellulären Komponenten untersucht. In dieser Arbeit konnte gezeigt werden, dass beide ICL3-Splicevarianten natürlicherweise in einem definierten Verhältnis auftreten. Außerdem konnte gezeigt werden, dass die lange ICL3-Variante des BAI2 nicht zu einer Änderung der Membrantopologie des Rezeptors, einer Homodimerisierung über die zusätzliche Aminosäuresequenz oder zu einer Interaktion mit dem C-Terminus führt. Die Splicevariante im humanen C-Terminus des BAI2 konnte als eine variable, durch Exonskipping entstandene Calcium-unabhängige Calmodulin-Bindungsstelle identifiziert werden. Diese Arbeit belegt die Existenz mehrerer BAI2-Isoformen in vivo. Die Struktur dieser Isoformen lässt unterschiedliche Funktionalitäten vermuten. Auch wenn erste Untersuchungen zwischen den beiden ICL3-Varianten keinen Unterschied ergaben, sind diese Erkenntnisse für die weitere Analyse der Signaltransduktion und Ligandensuche bedeutend. Es ist z.B. denkbar, dass sich die beiden ICL3-Varianten in der G-Protein-Kopplung oder bei der Rekrutierung von intrazellulären Interaktionspartnern unterscheiden oder dass die Splicevariante im C-Terminus zu einer Scaffold- Funktion des Calmodulins führt und/oder die Signaltransduktion durch eine permanente Bindung des Calmodulins an einer Isoform moduliert wird.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on August 28, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): William Bosron. Includes vita. Includes bibliographical references (leaves 102-111).

Abstract The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant. We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis. The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues. The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment. Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates.

The P2X7R is a functionally distinct member of the P2X non-selective cation channels and has been implicated in the initiation of immune responses. One of the most extensively characterised immune responses of the receptor is to signal the rapid aggregation of the inflammasome complex and signal the release of IL-1β. These investigations have focused in providing direct comparisons of P2X7R-driven IL-1 responses between DC and mouse macrophages (peritoneal macrophages [PMΦ] and bone marrow derived macrophages [BM-MΦ]). Expression of the P2X7R has been identified in all three populations both at the transcriptional (P2X7A variant) and protein levels. Activation with lipopolysaccharide (LPS) (2h) induced a rapid dose dependent release of IL-6 but not of IL-1β in BM-DC. Rapid (2h) IL-1β release required both LPS priming and ATP activation. Both signals were also required for IL- 1β release in mouse ΒΜ-ΜΦ and PMΦ, however, at comparatively markedly lower levels. Furthermore, like with IL-1β, LPS did not induce IL-1α release in BM-DC. Interestingly, subsequent challenge with ATP evoked IL-1α release in BM-DC alone, with little or no detectable levels observed in activated BM-MΦ. This rapid IL-1β release (but not IL-6) was potently inhibited in both macrophages and DC with a P2X7R-specific inhibitor (A-740003) providing evidence that is predominantly a P2X7R-driven process. Treatment with A-740003 also potently inhibited IL-1α release from BM-DC suggesting that the ATP-P2X7R and caspase-1 activation might have a role in the release of the cytokine. Expression of gain-of-function P2X7K and loss-of- function P2X7J splice variants has been identified in both BM-DC and BM-MΦ, at the level of transcription. The possibility that a differential baseline or LPS-induced expression (at the transcriptional level) of P2X7J and P2X7K variants accounts for the diverse cytokine responses observed in BM-DC and BM-MΦ was also explored. However, the levels of expression for the various splice variants of interest (P2X7K and P2X7J) were found to be similar between the two cell types. The results of these investigations identify some subtle but intriguing differences in the mechanism of P2X7R activation and IL-1 release between DCs and macrophages. Purinergic signalling is increasing being implicated in the regulation of immune responses both in potentiating or suppressing inflammation. However, further work is required to decipher how the dynamic interplay between different purines can influence the immune activation of different cell types and indeed different cell subsets.

The anteroventral periventricular nucleus (AVPV) is a sexually dimorphic preoptic structure that is nearly three times larger in the female rat. Estradiol (E2) exposure during development decreases AVPV volume through apoptosis, a process which normally occurs preferentially in male. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupter that interferes with E2-dependent sexual differentiation of the AVPV. Whole-genome microarrays were used to identify sex-specific genes regulated by E2 and TCDD in postnatal day 2 (P2) AVPV punches from untreated males and females, males treated with TCDD or vehicle, and females treated with E2 or vehicle. Trio emerged as a gene target regulated by E2 and TCDD, and this gene is essential for neurite outgrowth which is sexually dimorphic in the AVPV. My goal was to verify microarray data that Trio was expressed in the P2 AVPV and to test whether expression was affected by sex, E2 and TCDD. The microarray did not discriminate among Trio splice variants, 9S, 9L and Duet, so I mapped each of these in the P2 AVPV using in situ hybridization. I used quantitative real-time PCR to examine the effects of sex, E2 and TCDD on mRNA levels encoding each splice variant. I found that the AVPV expressed 9S and 9L at moderate levels and Duet at low levels. The expression of 9S and 9L mRNA was increased by E2 in females. However, only 9S expression differed between sexes and it was lower in males. TCDD had no effect on expression of any of the splice variants. This work provides the first evidence that Trio splice variants are independently regulated and that Trio may mediate effects of E2 in the developing AVPV.

No description available.

Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.

<p>Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.</p>

No description available.

No description available.