GRK2 wird an Serin29 durch PKC phosphoryliert. Die Phosphorylierung verhindert die Inhibition der GRK2 durch Calmodulin. Die Inhibition der GRK2 durch Calmodulin wird durch den N-Terminus der GRK2 vermittelt und ist auf eine gestörte Aktivierbarkeit der GRK2 durch G-Protein beta/gamma-Untereinheiten zurückzuführen. / GRK2 is phosphorylated by PKC at serin29. The phosphorylation prevents GRK2 inhibition by calmodulin. Inhibition of GRK2 by calmodulin is mediated by the N-terminus of the kinase and is due to a disturbed activation of GRK2 by G-protein beta/gamma subunits.

Universiẗat, Diss., 2005--Würzburg.

Parathormon (PTH) aktiviert am PTH-1 Rezeptor (P1R) mindestens zwei Signalwege: Über Guanosintriphosphat-bindende Proteine (Gs) kommt es intrazellulär zu einem Anstieg von zyklischem Adenosinmonophosphat (cAMP) und zu einer Aktivierung der Proteinkinase A (PKA). Gq Protein-vermittelt erfolgt eine Aktivierung der Phospholipase C (PLC) und ein intrazellulärer Anstieg des Inositoltriphosphat (IP3). Der verwandte PTH-2 Rezeptor (P2R) wird einerseits durch seinen vermutlich physiologischen Liganden, das tuberoinfundibuläre Peptid (TIP39), und andererseits durch PTH aktiviert. Im Gegensatz zum P1R zeigt der P2R nur eine Ankopplung an den cAMP Signalweg und keine an den PLC Signalweg. Voruntersuchungen hatten gezeigt, dass intrazelluläre Abschnitte der zweiten und dritten Schleife sowie Bereiche des C-Terminus des P1R für die Aktivierung des PLC Signalweges verantwortlich sind. In der vorliegenden Dissertation erfolgte eine stufenweise Angleichung intrazellulärer Abschnitte des P2R an den P1R. Die generierten Hybridrezeptoren wurden hinsichtlich ihres Signaltransduktionsverhaltens untersucht. Mit der Bestimmung der akkumulierten Gesamtinositole und des intrazellulären Calciums [Ca]i konnte gezeigt werden, dass trotz einer 95%-igen Übereinstimmung der intrazellulären Aminosäuresequenzen der P2R-Hybridrezeptoren mit denen des P1R, die Ankopplung an den PLC Signalweg nicht übertragen werden kann. Diese Ergebnisse wurden für Stimulationen mit PTH und TIP39 nachgewiesen. Durch Hemmung der Proteinkinasen A und C konnte, wie erwartet, am P1R ein verstärktes IP3 Signal beobachtet werden. Eine Aktivierbarkeit des PLC Signalweges wurde auch hierbei für die Hybridrezeptoren nicht gesehen. Für die Aktivierung des cAMP Signalweges der Hybridrezeptoren konnte beobachtet werden, dass diese sich weitestgehend in Anlehnung zum P2R verhalten. Außerdem konnte gezeigt werden, dass für eine effiziente Ankopplung an den cAMP Signalweg alle aus dem P1R in den P2R einfügten Teilabschnitte (zweite, dritte Schleife und C-Terminus) zusammenwirken. Der Hybridrezeptor, an dem alle drei Teilabschnitte ausgetauscht wurden, führte zu einer signifikant besseren Ankopplung an den cAMP Signalweg, als die Einzelmutation des C-Terminus. Die Messung zum cAMP Signalweg wurden mit den Liganden TIP39 und PTH durchgeführt. Für die Aktivierung des Gs Protein-vermittelten cAMP Signalweg am P1R oder P2R sind daher vermutlich Interaktionen verschiedener Rezeptorabschnitte verantwortlich. Insgesamt konnten in dieser Arbeit weitere wichtige Erkenntnisse bezüglich unterschiedlicher Aspekte der Signaltransduktion des P1R und des P2R gewonnen werden. / Activation of the phospholipase C (PLC) pathway upon stimulation with PTH is unique to the PTH-1 receptor (P1R) and is virtually absent in the PTH-2 receptor (P2R). The sites of P1R mutations known to reduce PLC signaling may achieve this indirectly by disturbing coupling at other sites. We therefore chose a more stringent approach and attempt to reestablish PLC signaling in the closely (70%) related P2R by gradually adapting the P2Rs cytosolic interface to a P1R-like sequence by engineering hybrid P1R/P2R constructs. Key differing regions of the P1R´s second loop (DT272-273:EK), third loop (IW344-345:LR), and C-terminal tail (T440-end: K-end) were put into the P2R. A fourth chimeric P1R/P2R receptor, combining all these changes, thus had a > 90% sequence identity with the cytosolic interface of the P1R. All mutants were expressed on the cell surface of stably transfected HEK 293 cells, using a radioreceptor assay with [125I]-Nle8,21-Tyr34-rat PTH(1-34), with a similar density of 1.1 to 2.5 mio receptors/cell. Activation of the PLC pathway was assessed by: 1) accumulation of total inositol phosphates in myo[3H]-inositol-prelabeled cells. No increase was detectable in the P2R or any of the receptor chimeras, even after enhancing the possible response by blockade of protein kinases A and C, despite an up to 13-fold increase with the P1R upon PTH stimulation. 2) Similarly, a PTH-induced increase in intracellular calcium (detected by fluo-3) of the wildtype P1R with as little as 1 nM hPTH(1-34) was completely absent in all mutants as well as in the P2R. The PTH-induced max. response of the cyclic AMP pathway was virtually identical for the P1R and P2R. However, the max. response of the mutant with the P1R tail sequence was reduced to only 35% (PTH) and 26% (TIP39) of the P2R response. Surprisingly, this signaling loss could be overcome by introducing more P1R sequence into the second and third cytoplasmic loop of this P1R/P2R hybrid receptor thus regaining a max. cAMP response of 64% (PTH) and 85% (TIP39). Presumably, an interaction of P1R sequences seems to result in a more effective coupling to the cAMP signaling pathway. In conclusion, these results suggest that the activation of the IP / intracellular calcium signaling pathway by the P1R is highly dependent on a P1R-like intracellular contact interface, which can not be mimicked easily even in the presence of > 90% of P1R sequence in the cytosolic interface of the P2R.

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.