The study of arrestins as regulators of seven transmembrane receptor (7TMR) signaling has revealed multiple levels of complexity, initiating desensitization of G protein activity and coordination of receptor internalization via clathrin‐coated pits. Recently, β‐arrestins have also been shown to act as adaptor proteins, mediating G protein‐independent signaling as well as scaffolding of enzymes that degrade second messenger molecules. This latter function was demonstrated by β‐arrestins recruiting PDE4 phosphodiesterase to Gs‐coupled β2‐adrenergic receptors, enhancing metabolism of the second messenger cAMP. As β‐arrestins universally interact with members of the 7TMR superfamily, we sought to determine if this phenomenon of concerted desensitization might be applicable to additional receptor subtypes. We screened for β‐arrestin‐binding proteins among modulators of diacylglycerol and IP3 (second messengers downstream of Gq‐coupled 7TMRs). We observed β‐ arrestins constitutively interacted with members of the diacylglycerol kinase (DGK) family, which phosphorylate diacylglycerol to create phosphatidic acid. Furthermore, examining lipid extracts of 32P labeled cells separated by TLC, we observed that overexpression of β‐arrestin enhanced phosphatidic acid (PA) production after M1 muscarinic receptor stimulation. Conversely, depletion of β‐arrestins by RNA interference showed significantly decreased agonist‐stimulated PA accumulation. Additionally, overexpression of a β‐arrestin2 mutant that binds DGKs but not receptors served as a dominant negative for agonist‐dependent DGK activity. These results demonstrate a requirement for β‐arrestins in DGK translocation to the membrane, and specifically to activated 7TMRs, where concentrations of second messengers are at their highest. Phosphatidic acid is an effector for several enzymes, including the phosphatidylinositol 5‐kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus, we hypothesized β‐arrestin‐targeted DGKs may regulate PIP5K activity. PIP5K Iα associated with β‐arrestin2 in an agonist‐dependent manner in HEK293 cells, and a β‐ arrestin2 mutant defective in receptor endocytosis (a PIP2‐dependent function) was impaired. Furthermore, knockdown of β‐arrestin2 by RNAi significantly decreased the amount of PIP5K Iα detected in receptor immunoprecipitates. In TLC assays, overexpressing both β‐arrestin2 and PIP5K Iα enhanced agonist‐stimulated PIP2 labeling, while either protein alone had no effect. These data support the concept of β‐ arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which then stimulates production of PIP2, promoting receptor internalization. / Dissertation

This thesis describes the expression of three human seven transmembrane receptors: the M2 Muscarinic; H1 Histamine and 5HT2A Serotonin receptors, in the baculovirus/insect cell expression system. Purification trials werre conducted on the M2 Muscarinic and H1 Histamine receptors. Preliminary crystallisation attempts were made with the H1 receptor.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13146号 / 論農博第2852号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5093(農学部図書室) / (主査)教授 植田 和光, 教授 加納 健司, 教授 三芳 秀人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Fibronectin is an extracellular multidomain glycoprotein that directs and regulates a variety of cell processes such as proliferation, development, haemostasis, embryogenesis, and wound healing. As a major component of blood, fibronectin exists as a soluble disulphide linked dimer, but it can also be incorporated into an insoluble cross-linked fibrillar network to form a major component of the extracellular matrix. Fibronectin is composed of an extended chain of module repeats termed Fn1, Fn2, and Fn3 that bind to a wide range of transmembrane receptors and extracellular matrix components, including collagen. The gelatin binding domain of fibronectin was first isolated as a 45kDa proteolytic fragment and has since been found to be composed of six modules: 6Fn1-1Fn2-2Fn2-7Fn1-8Fn1-9Fn1 (in this notation nFX represents the nth type X module in the native protein). This domain has been reported to bind to both collagen and denatured collagen (gelatin), but with 10-100 times higher affinity to the latter; it can be purified to homogeneity on a gelatin affinity column. In the work presented here, fragments of the gelatin binding domain are expressed in P. pastoris, purified to homogeneity, and investigated at the molecular level. Through a dissection approach, surface plasmon resonance (SPR) is used to characterise the recombinantly produced protein, to accumulate more information about the function of the full domain. NMR is used to assess the folding of the protein fragments at atomic resolution. In particular, the secondary structure of 8Fn1-9Fn1 is mapped using inter-strand NOEs, which suggests that the construct takes the fold of a pair of typical Fn1 modules. Gelatin affinity chromatography is used to confirm that both Fn1 and Fn2 modules contribute to gelatin binding, possibly in two clusters (1Fn2-2Fn2 and 8Fn1-9Fn1). The 7Fn1 module may perform a structural role in linking together these two interaction sites, in the same way as suggested for 6Fn1, which is thought to act in a structural manner to enhance the binding of 1Fn2-2Fn2 to gelatin. Three carbohydrate moieties are found on this domain, one on 2Fn2 and two on 8Fn1. Here, by means of expressing different protein length fragments, and by site directed mutagenesis, the role of each sugar chain is investigated independently. The sugar chain on 2Fn2 does not appear to promote binding to collagen, nor does the first sugar chain on 8Fn1 (N-linked to N497), implying another role for these sugars such as protection from proteolysis. However, the presence of at least a single GlcNAc sugar residue on the second sugar chain site on 8Fn1 (N- linked to N511) is essential for full affinity binding to collagen. Direct binding of the 8Fn1-9Fn1 module pair to collagen is assessed with a short collagen peptide and the binding is monitored by NMR. The peptide appears to bind, predominantly to the final strand of 8Fn1, the first β- strand of 9Fn1, and the linker between the two modules, with μM affinity. A model for bound peptide is proposed. The highly conserved amino acid motif Ile-Gly-Asp (IGD) is found on four of the nine N-terminal Fn1 modules of fibronectin. Tetrapeptides containing the IGD were demonstrated to promote the migration of fibroblast cells into a native collagen matrix. Two of these “bioactive” IGD motifs are found within the gelatin binding domain, one on 7Fn1 and one on 9Fn1. In this study, the motif in the 8Fn1-9Fn1 module pair is shown to be located in a tightly constrained loop within 9Fn1. By site directed mutagenesis, the IGD motifs of 7Fn1 and 9Fn1 are subjected to single amino acid substitutions, and their ability to stimulate cell migration assessed in our assay. By NMR, the fold of the IGD mutant proteins is found to be unaffected by the mutation with respect to the wild type, with the exception of small perturbations around the substitution site. While the wild type module is able to stimulate fibroblast migration, the mutant proteins show reduced or negligible bioactivity. The larger fragments show far more potency in stimulating fibroblast migration, with 8Fn1-9Fn1 (one IGD motif) 104 times more potent than the IGD peptide, and the full gelatin binding domain (two IGD motifs) 106 times more potent than the 8Fn1-9Fn1. Potential mechanisms for this enormous enhancement of the IGD potency in different contexts are discussed.