Transport of cell adhesion molecules: How does Rab21 organise integrin trafficking on a mechanistic level?

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Numerous diseases such as cancer progression, inflammation and degenerative disorders are results of deregulated cell adhesion and migration. The integrin family of transmembrane cell surface receptors have a decisive impact on the migratory and invasive capacities of cancer and immune cells. Furthermore, a variety of pathogens use integrins to invade host cells for infection, extending the relevance of integrins to infectious diseases. It is becoming increasingly evident that not only activation/deactivation, but also the endo-/exocytic cycle of integrins plays an important role in mediating integrin function. The small Ras-family GTP-binding protein Rab21, which binds to the cytoplasmic tail of alpha-integrins, is a major regulator in the endocytic trafficking of integrins. However, on a molecular or structural level the mode of action remains poorly understood mainly due to the lack of known Rab21-interacting proteins.The aim of the proposed research is to gain insights how Rab21 organises integrin trafficking on a structural-mechanistic level. To answer this question, three projects will be implemented.A) Identification of Rab21 effector proteins involved in integrin trafficking.Novel Rab21 downstream effector proteins will be identified using a quantitative mass spectrometry-based protein-protein interaction screen and tested regarding their influence on integrin trafficking.B) Structural analysis of the Rab21-integrin trafficking network. After their identification, Rab21 downstream effectors as well as the cytoplasmic tail of alpha-integrins will be crystallised in complexes with Rab21 to obtain a structural snapshot of the Rab21-regulated integrin trafficking network. The X-ray structures will provide insights into the function of the trafficking complex and answer the question how Rab21 manages to transport cargo (integrin) and bind effector proteins in parallel.C) Functional analysis of the Rab21 effector proteins. I will investigate the biochemical and cell biological function of the identified effector proteins and address the question how these effector proteins effect the dynamics of integrin endocytosis. Different microscopy techniques (confocal microscopy, live cell imaging, TIRF) will be employed.In this proposed research, I will employ a multidisciplinary approach starting with a biochemical screen and combining various state of the art and cutting edge cell and structural biological tools to maximise our mechanistic understanding of the question how integrins are trafficking from the plasma membrane to endosomes and how this trafficking influences different disease processes.