Structural and Functional Studies of Gas6

Abstract: The vitamin K-dependent and ubiquitously expressed 82 kDa glycoprotein product of the growth-arrest-specific-gene 6, Gas6, shows 44% sequence identity to the anticoagulant protein, protein S. Both proteins share further the same domain organization. A short loop region, 4 epidermal growth factor-like modules and an SHBG-like region follow the N-terminal Gla-domain, which confers membrane-binding capability. The Gas6 SHBG interacts with the receptor tyrosine kinases of the Tyro3 subfamily, Axl, Tyro3 and Mer. As a ligand to these receptors Gas6 is implicated in mediating for example growth, survival, migration, clearance of apoptotic cells and platelet response amplification. The investigations described in this thesis were aimed at shedding further light on the structure and function of these relatively recently discovered proteins. In study I, purified Gas6 was added to Gas6 deficient mouse cardiac fibroblasts, which responded by mitogenesis and increased survival. Employing a sensitive ELISA, in study II, Gas6 could be detected in human circulation (? 18 ng/mL) but not in human platelets. In study III it's shown that most of the Gas6 present in human circulation likely is complex bound to soluble extracellular domains of Axl. We further show that Gas6 ability to interact with its receptors relies on calcium. Study IV shows the high tendency of Gas6 to form multimers during purification. The multimers, which fail to interact with Axl, show slow association but high affinity binding towards bilayers containing phosphatidylserine. By contrast the monomers display fast association, low affinity binding and preference for bilayers containing phosphatidylethanolamine in addition to phosphatidylserine.