Extracellular matrix promotes clathrin-dependent endocytosis of prolactin and STAT5 activation in differentiating mammary epithelial cells.

The hormone prolactin promotes lactational differentiation of mammary epithelial cells (MECs) via its cognate receptor and the downstream JAK2-STAT5a signalling pathway. In turn this regulates transcription of milk protein genes. Prolactin signalling depends on a cross-talk with basement membrane extracellular matrix (ECM) via β1 integrins which activate both ILK and Rac1 and are required for STAT5a activation and lactational differentiation. Endocytosis is an important regulator of signalling. It can both enhance and suppress cytokine signalling, although the role of endocytosis for prolactin signalling is not known. Here we show that clathrin-mediated endocytosis is required for ECM-dependent STAT5 activation. In the presence of ECM, prolactin is internalised via a clathrin-dependent, but caveolin-independent, route. This occurs independently from JAK2 and Rac signalling, but is required for full phosphorylation and activation of STAT5. Prolactin is internalised into early endosomes, where the master early endosome regulator Rab5b promotes STAT5 phosphorylation. These data reveal a novel role for ECM-driven endocytosis in the positive regulation of cytokine signalling.