Principal Investigator: Herbert M. Geller
Grant Title: Novel Glycosaminoglycan Binding to Type IIa Protein Phosphatase
PROGRESS REPORT for Mizutani Foundation Research Grant
Reference Number: 17-0116
Principal Investigator: Herbert M. Geller
Organization: National Heart, Lung, and Blood Institute
Period: From April 2017 to March 2018
Grant Title: Novel Glycosaminoglycan Binding to Type IIa Protein Phosphatases

Abstract
Emerging evidence suggests that proteoglycans are independent signaling molecules,
binding directly to transmembrane receptors to exert distinct biological effects. In thenervous system, the glycosaminoglycan (GAG) chains of proteoglycans bind to membersof the Type IIa receptor protein tyrosine phosphatases (RPTPσ/RPTPδ/LAR). RPTPσ iscomposed of two cytoplasmic phosphatase domains and extracellular immunoglobulin (Ig)domains followed by fibronectin type III (FNIII) repeats (Figure). Previous mutagenesis
and structural studies have shown that a GAG-binding site for both heparan sulfate (HS)and chondroitin sulfate (CS) lies in the first Ig domain of RPTPσ and comprises anextended positively charged surface of basic residues (Lys- and Arg-loop). This has led tothe conundrum that both HS and CS bind to the same site but produce opposite actions:
HS promotes axonal growth and CS impedes the growth of axons.
We have investigated whether the complex formation between full length RPTPσand GAGs impacts the receptor’s enzymatic activity under physiological conditions.
When 293 cells expressing wild-type RPTPσ were treated with heparin, a mimic of HS,
the level of Tyr phosphorylation (pTyr) was transiently upregulated. Intriguingly, theincrease in pTyr by heparin was found even after disruption of the Lys/Arg loop (∆Lysmutation) whereas CS-E-induced upregulation was substantially reduced by the mutation.
Solid-phase binding assays with the extracellular domain (ECD) of RPTPσ showed thepresence of multiple binding sites for heparin on RPTPσ. The fact that heparin binding
upregulated pTyr independently of Lys/Arg loop binding site on RPTPσ led us to identify
novel heparin binding sites on the receptor.
To further characterize the novel binding site(s), a “heparin protection assay” wasperformed to identify potential amino acids, followed by mutagenesis of these amino acids
together with heparin affinity chromatography. ThisIg domain:
CSPGFNIII domain:
allowed the identification of 2 novel heparin-binding
Phosphatasedomain:sites located in the juxtamembrane domain (Figure).
HS GAG:CS GAG:
Disruption of all 3 heparin-binding sites (one in the 1stSulfation:
RPTPσtransIg domain and two in the juxtamembrane domain)
completely eliminated the binding to heparin.
HSPGNovel Heparin
One question that arises here is whether theseBinding Sites
cisnovel heparin-binding sites are involved in the alteredTM
phosphorylation upon heparin binding. When theAltered
Alteredheparin-binding deficient mutant was expressed onPhosphorylation
Phosphorylation
293 cells, we observed very high level of baseline
Inhibitneurite outgrowth1

Promoteneurite outgrowthPrincipal Investigator: Herbert M. Geller
Grant Title: Novel Glycosaminoglycan Binding to Type IIa Protein Phosphatase
pTyr without heparin. Addition of heparin did not alter the level of phosphorylation. Thisimplies that elimination of all heparin binding sites on RPTPσ caused the dysregulation ofpTyr within the cells. Thus, we propose a model for differential interactions of HS and CS
GAG chains with RPTPσ (Figure). HSPGs on the same cell surface interact in cis withRPTPσ through the novel binding sites and regulate the enzymatic activity of RPTPσ.
When CSPGs are presented in trans, the binding is through the first Ig domain andtriggers the changes in phosphorylation of distinct sets of proteins within the cells,
resulting in the recognition of CS as inhibitory cues by RPTPσ. Our discovery of new
heparin binding sites near the transmembrane domain of RP provides new insights intoGAG signaling.