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CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

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cPTPRO protein is developmentally regulated in embryonic chick brain. 20 μg of brain P2 fractions from E5, E7, E10, E14, E18, and E21 was separated by SDS-PAGE and subjected to Western blot analysis using affinity-purified anti-cPTPRO antibody. (A) cPTPRO migrated with an apparent molecular mass of 180 kD (arrow). Note the increase in expression from E5 to E10 and the decrease after E14. (B) Quantitative analysis from three independent experiments. Data were plotted as a percentage of the levels at E10 ± SEM.
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fig1: cPTPRO protein is developmentally regulated in embryonic chick brain. 20 μg of brain P2 fractions from E5, E7, E10, E14, E18, and E21 was separated by SDS-PAGE and subjected to Western blot analysis using affinity-purified anti-cPTPRO antibody. (A) cPTPRO migrated with an apparent molecular mass of 180 kD (arrow). Note the increase in expression from E5 to E10 and the decrease after E14. (B) Quantitative analysis from three independent experiments. Data were plotted as a percentage of the levels at E10 ± SEM.

Mentions: cPTPRO mRNA is upregulated between embryonic days 8 (E8) and 13 (E13) in chick brain, coinciding with the major period of axon outgrowth (Bodden and Bixby, 1996). To determine if cPTPRO protein expression follows this pattern, we used an antibody to a fusion protein from the ECD of cPTPRO (Ledig et al., 1999b) to probe Western blots of membrane fractions from chick brain. This antibody recognized a specific cPTPRO band at 180 kD that was not present in membranes from liver or muscle (unpublished data). This size is similar to that seen for cPTPRO orthologues in other species (Tagawa et al., 1994; Thomas et al., 1994; Wiggins et al., 1995), and is the size encoded by the full-length cDNA when expressed in transfected COS cells (unpublished data). As the predicted protein encoded by the PTPRO cDNA is 141 kD (Bodden and Bixby, 1996), our data are consistent with a high degree of glycosylation. PTPRO expression in brain was low at E5, increased dramatically between E7 and E10, then declined after E14, consistent with our previous findings for mRNA expression (Fig. 1) . This pattern is similar to that suggested by immunofluorescence experiments in retina and tectum, and is consistent with our hypothesis that cPTPRO plays a role in axon outgrowth during development.


CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

cPTPRO protein is developmentally regulated in embryonic chick brain. 20 μg of brain P2 fractions from E5, E7, E10, E14, E18, and E21 was separated by SDS-PAGE and subjected to Western blot analysis using affinity-purified anti-cPTPRO antibody. (A) cPTPRO migrated with an apparent molecular mass of 180 kD (arrow). Note the increase in expression from E5 to E10 and the decrease after E14. (B) Quantitative analysis from three independent experiments. Data were plotted as a percentage of the levels at E10 ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196468&req=5

fig1: cPTPRO protein is developmentally regulated in embryonic chick brain. 20 μg of brain P2 fractions from E5, E7, E10, E14, E18, and E21 was separated by SDS-PAGE and subjected to Western blot analysis using affinity-purified anti-cPTPRO antibody. (A) cPTPRO migrated with an apparent molecular mass of 180 kD (arrow). Note the increase in expression from E5 to E10 and the decrease after E14. (B) Quantitative analysis from three independent experiments. Data were plotted as a percentage of the levels at E10 ± SEM.
Mentions: cPTPRO mRNA is upregulated between embryonic days 8 (E8) and 13 (E13) in chick brain, coinciding with the major period of axon outgrowth (Bodden and Bixby, 1996). To determine if cPTPRO protein expression follows this pattern, we used an antibody to a fusion protein from the ECD of cPTPRO (Ledig et al., 1999b) to probe Western blots of membrane fractions from chick brain. This antibody recognized a specific cPTPRO band at 180 kD that was not present in membranes from liver or muscle (unpublished data). This size is similar to that seen for cPTPRO orthologues in other species (Tagawa et al., 1994; Thomas et al., 1994; Wiggins et al., 1995), and is the size encoded by the full-length cDNA when expressed in transfected COS cells (unpublished data). As the predicted protein encoded by the PTPRO cDNA is 141 kD (Bodden and Bixby, 1996), our data are consistent with a high degree of glycosylation. PTPRO expression in brain was low at E5, increased dramatically between E7 and E10, then declined after E14, consistent with our previous findings for mRNA expression (Fig. 1) . This pattern is similar to that suggested by immunofluorescence experiments in retina and tectum, and is consistent with our hypothesis that cPTPRO plays a role in axon outgrowth during development.

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

Show MeSH
Related in: MedlinePlus