title: MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.
abstract: The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.
authors: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors: Shameka J
authors: SJ
authors: Shelby
authors: Karen
authors: K
authors: Colwill
authors: Sirano
authors: S
authors: Dhe-Paganon
authors: Tony
authors: T
authors: Pawson
authors: Debra A
authors: DA
authors: Thompson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002829:Choroid / Q000378:metabolism; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010587:Phagocytosis / Q000235:genetics; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015398:Signal Transduction; D000076202:c-Mer Tyrosine Kinase; D018909:src Homology Domains; D019061:src-Family Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011993:Recombinant Fusion Proteins; C550255:VAV3 protein, rat; D019000:Phosphotyrosine; D058543:Class Ia Phosphatidylinositol 3-Kinase; C097072:MERTK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase; D019061:src-Family Kinases
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