Abstract
BackgroundThe human POB1/REPS2 (Partner of RalBP1) protein is highly conserved in mammals where it has been suggested to function as a molecular scaffold recruiting proteins involved in vesicular traffic and linking them to the actin cytoskeleton remodeling machinery. More recently POB1/REPS2 was found highly expressed in androgen-dependent prostate cancer cell lines, while one of its isoforms (isoform 2) is down regulated during prostate cancer progression.ResultsIn this report we characterize the central proline rich domain of POB1/REPS2 and we describe for the first time its functional role in receptor endocytosis. We show that the ectopic expression of this domain has a dominant negative effect on the endocytosis of activated epidermal growth factor receptor (EGFR) while leaving transferrin receptor endocytosis unaffected. By a combination of different approaches (phage display, bioinformatics predictions, peptide arrays, mutagenic analysis, in vivo co-immunoprecipitation), we have identified two closely spaced binding motifs for 14-3-3 and for the SH3 of the proteins Amphiphysin II and Grb2. Differently from wild type, proline rich domains that are altered in these motifs do not inhibit EGFR endocytosis, suggesting that these binding motifs play a functional role in this process.ConclusionOur findings are relevant to the characterization of the molecular mechanism underlying the involvement of POB1/REPS2, SH3 and 14-3-3 proteins in receptor endocytosis, suggesting that 14-3-3 could work by bridging the EGF receptor and the scaffold protein POB1/REPS2.
Highlights
The human POB1/REPS2 (Partner of RalBP1) protein is highly conserved in mammals where it has been suggested to function as a molecular scaffold recruiting proteins involved in vesicular traffic and linking them to the actin cytoskeleton remodeling machinery
We show that, in addition to the amino-terminal EH domain and the carboxy-terminal RALBP1 binding region, a third central area of POB1, containing motifs recognized by the Src homology 3 domain (SH3) domains of Grb2 and Amphiphysin II and by the 14-3-3 family proteins, plays a role in modulating receptor endocytosis
We were interested in assessing whether the POB1 proline-rich region plays an additional role in the ligand-dependent endocytosis of epidermal growth factor receptor (EGFR)
Summary
The human POB1/REPS2 (Partner of RalBP1) protein is highly conserved in mammals where it has been suggested to function as a molecular scaffold recruiting proteins involved in vesicular traffic and linking them to the actin cytoskeleton remodeling machinery. More recently POB1/REPS2 was found highly expressed in androgen-dependent prostate cancer cell lines, while one of its isoforms (isoform 2) is down regulated during prostate cancer progression. The observed interactions with proteins involved in the endocytic pathway and the report that over-expression of the EH domain or the RalBP1 binding region inhibits EGF and insulin internalization, implicates POB1 in receptor endocytosis [4]. POB1 coimmunoprecipitates with over expressed Grb (growth factor receptor-bound protein 2) and with PAG2 (DDEF1), a paxillin-associated protein, at endogenous levels. The sequence responsible for this latter interaction was mapped to a proline-rich motif (423PSKPIR428) that binds to the SH3 domain of PAG2 [6]
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