Fig. 2. The role of the cytosolic domain in the internalisation of CD317. (A,B) COS cells were transiently transfected with constructs encoding either full-length, truncated or mutated CD317 as indicated and subjected to anti-CD317 antibody uptake for 15 minutes. CD317-antibody uptake is clearly inhibited in cells expressing either truncated CD317 (MDER panel) or CD317 with both Y6A and Y8A mutations (MAPSFAHA panel), as shown by the accumulation of antibody at the plasma membrane in these cells. By contrast, the punctate intracellular fluorescent signal shows that there is efficient internalization of CD317 in cells expressing either the Y6A (MAPSFAHY) or Y8A (MAPSFYHA) mutations. The cytosolic N-terminal sequence of full-length rat CD317 is MAPSFYHYLPVAMDER. The MDER construct lacks the N-terminal 12 amino acids of the cytosolic domain, the MAPSFAHY construct has a Y6A mutation, the MAPSFYHA construct has a Y8A mutation and the MAPSFAHA construct has the point mutations Y6A and Y8A. All mutations are in the context of the full-length cytosolic domain. Bar, 10 µm. ID, intracellular domain; TM, transmembrane domain; ED, extracellular domain and GPI; glycosylphosphatidylinositol anchor.