Regulation by Dimerization

Dimerization plays a critical role in the regulation of another family of transmembrane proteins, the receptor tyro-sine kinases. Specifically, ligand binding to the extracellular domain allows the intracellular kinase domain to dimerize and cross-phosphorylate at regulatory sites, leading to activation of the intracellular kinase domain. Two independent crystal structures of the membrane-proximal phosphatase domain, D1, of RPTPa reveal a symmetrical dimer in which the active site of one domain is occluded by a helix-turn-helix wedge of its dimer forming partner [1]. Disulfide-bonding experiments demonstrate that this dimeric configuration renders RPTPa catalytically inactive in vivo [2]. Since the initial RPTPa crystal structure was revealed, a plethora of evidence has surfaced supporting the idea that dimerization is an important regulatory tool in RPTPs. Most notably, Tertoolen et al. [3] used fluorescence resonance energy transfer to demonstrate that RPTPa dimerizes constitutively in living cells and that the transmembrane region is sufficient for dimer formation. RPTPa dimerization is a negative regulatory event, in contrast to activation of receptor tyrosine kinases by dimerization (see Fig. 1A). Dimerization also plays a crucial role in the regulation of another RPTP family member, CD45. Recombinant D1 from CD45 exists primarily as a dimer as assessed by gel filtration chromatography [4]. EGF-enhanced dimerization of the CD45 intracellular domain linked to the extracellular ligand binding and transmembrane domain of EGFR results in CD45 inactivation consistent with a regulatory model in which dimerization serves as a negative regulatory signal [5]. Like RPTPa, CD45 dimerization is dependent on the wedge region located in the membrane-proximal D1 phosphatase domain. A knock-in mutant mouse containing a point mutation in the wedge region of CD45 (Glu613 to Arg) that inhibits dimer formation

Inactive Form Active Form GRB2 Bound Form cSRC Bound Form

Figure 1 (A) The membrane-proximal phosphatase domain, D1, of RPTPa forms an inhibited dimer in which the active site of one monomer is sterically occluded by a wedge region of its dimer partner; perhaps serine phosphorylation in the juxtamembrane region serves to regulate dimer formation. (B) The C-terminus of RPTPa is tyrosine phosphorylated, allowing binding of the SH2-domain-containing protein Grb-2. This binding does not result in the recruitment of associated factors but may mask the site to inhibit binding of the SH2 domain of cSRC. Under the proper conditions, cSRC can bind the pTyr C-terminus, allowing RPTPa to dephosphorylate and activate cSRC.

Inactive Form Active Form GRB2 Bound Form cSRC Bound Form

Figure 1 (A) The membrane-proximal phosphatase domain, D1, of RPTPa forms an inhibited dimer in which the active site of one monomer is sterically occluded by a wedge region of its dimer partner; perhaps serine phosphorylation in the juxtamembrane region serves to regulate dimer formation. (B) The C-terminus of RPTPa is tyrosine phosphorylated, allowing binding of the SH2-domain-containing protein Grb-2. This binding does not result in the recruitment of associated factors but may mask the site to inhibit binding of the SH2 domain of cSRC. Under the proper conditions, cSRC can bind the pTyr C-terminus, allowing RPTPa to dephosphorylate and activate cSRC.

exhibits a variety of phenotypes, including polyclonal lymphocyte activation consistent with an increase in cellular CD45 activity [6].

The dimerization model for RPTP regulation may not be a universal mechanism employed by all RPTPs. The crystal structures of the membrane-proximal phosphatase domain of RPTP^ and the tandem phosphatase domains of LAR failed to show dimer formation through the inhibitory wedge region [7,8]. Nevertheless, both structures contained an intact wedge that is not shared with cytosolic PTPs. It is important to note that the constructs used to crystallize both RPTP^ and LAR did not contain the transmembrane region that Tertoolen et al. demonstrated is sufficient for dimer formation in RPTPa [3]. It is unlikely that the wedge region provides sufficient binding energy to drive dimerization of RPTPa in vivo; therefore, additional regions such as the transmembrane segment are likely important for dimer formation [8A].

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