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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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Expression and purification of the cPTPRO–Fc fusion protein. (A) Schematic diagram of cPTPRO. The horizontal line represents the plasma membrane (TM), with extracellular area above. The ECD of cPTPRO consists of eight fibronectin type III repeats (▪). The intracellular portion contains one phosphatase domain (▭). (B) Diagram of the fusion protein. The protein is a disulfide-linked (S-S) dimer, with the ECD of cPTPRO fused to the hinge and constant regions (CH2, CH3) of mouse IgG-1. (C) 1 μg of purified cPTPRO–Fc was run on SDS-PAGE and stained with Coomassie blue. (D) 20 μg of cPTPRO–Fc was subjected to Western blot analysis with an anti-cPTPRO antibody. cPTPRO–Fc migrated with an apparent molecular mass of 170 kD under reducing (R) conditions and 350 kD under nonreducing (NR) conditions. Numbered arrows represent the molecular mass in kD of molecular weight standards.
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fig2: Expression and purification of the cPTPRO–Fc fusion protein. (A) Schematic diagram of cPTPRO. The horizontal line represents the plasma membrane (TM), with extracellular area above. The ECD of cPTPRO consists of eight fibronectin type III repeats (▪). The intracellular portion contains one phosphatase domain (▭). (B) Diagram of the fusion protein. The protein is a disulfide-linked (S-S) dimer, with the ECD of cPTPRO fused to the hinge and constant regions (CH2, CH3) of mouse IgG-1. (C) 1 μg of purified cPTPRO–Fc was run on SDS-PAGE and stained with Coomassie blue. (D) 20 μg of cPTPRO–Fc was subjected to Western blot analysis with an anti-cPTPRO antibody. cPTPRO–Fc migrated with an apparent molecular mass of 170 kD under reducing (R) conditions and 350 kD under nonreducing (NR) conditions. Numbered arrows represent the molecular mass in kD of molecular weight standards.

Mentions: Although nothing is known concerning ligand–receptor interactions of type III RPTPs, several type II RPTPs can bind homophilically, serving as both ligands and receptors (Bixby, 2000). Ligand–receptor interactions of these CAM-like RPTPs as well as those of other CAMs have been examined productively using Fc fusion proteins of the ECDs (Walsh and Doherty, 1997; Drosopoulos et al., 1999; Wang and Bixby, 1999). To investigate these interactions for cPTPRO, we fused the cDNA encoding the ECD of cPTPRO to the cDNA encoding the Fc domain of mouse IgG-1 (mIgG; Fig. 2, A and B) . This construct was used to express the fusion protein (cPTPRO–Fc) in stably transfected CHO cells, and purified with anti-mIgG agarose. As expected, the purified protein migrated as a 170-kD band under reducing conditions and as a 350-kD dimer under nonreducing conditions (Fig. 2 C). Bands of these same sizes were identified by an anti-cPTPRO antibody on Western blots of the purified proteins (Fig. 2 D), confirming the presence of the cPTPRO ECD in the fusion protein.


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)

Expression and purification of the cPTPRO–Fc fusion protein. (A) Schematic diagram of cPTPRO. The horizontal line represents the plasma membrane (TM), with extracellular area above. The ECD of cPTPRO consists of eight fibronectin type III repeats (▪). The intracellular portion contains one phosphatase domain (▭). (B) Diagram of the fusion protein. The protein is a disulfide-linked (S-S) dimer, with the ECD of cPTPRO fused to the hinge and constant regions (CH2, CH3) of mouse IgG-1. (C) 1 μg of purified cPTPRO–Fc was run on SDS-PAGE and stained with Coomassie blue. (D) 20 μg of cPTPRO–Fc was subjected to Western blot analysis with an anti-cPTPRO antibody. cPTPRO–Fc migrated with an apparent molecular mass of 170 kD under reducing (R) conditions and 350 kD under nonreducing (NR) conditions. Numbered arrows represent the molecular mass in kD of molecular weight standards.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196468&req=5

fig2: Expression and purification of the cPTPRO–Fc fusion protein. (A) Schematic diagram of cPTPRO. The horizontal line represents the plasma membrane (TM), with extracellular area above. The ECD of cPTPRO consists of eight fibronectin type III repeats (▪). The intracellular portion contains one phosphatase domain (▭). (B) Diagram of the fusion protein. The protein is a disulfide-linked (S-S) dimer, with the ECD of cPTPRO fused to the hinge and constant regions (CH2, CH3) of mouse IgG-1. (C) 1 μg of purified cPTPRO–Fc was run on SDS-PAGE and stained with Coomassie blue. (D) 20 μg of cPTPRO–Fc was subjected to Western blot analysis with an anti-cPTPRO antibody. cPTPRO–Fc migrated with an apparent molecular mass of 170 kD under reducing (R) conditions and 350 kD under nonreducing (NR) conditions. Numbered arrows represent the molecular mass in kD of molecular weight standards.
Mentions: Although nothing is known concerning ligand–receptor interactions of type III RPTPs, several type II RPTPs can bind homophilically, serving as both ligands and receptors (Bixby, 2000). Ligand–receptor interactions of these CAM-like RPTPs as well as those of other CAMs have been examined productively using Fc fusion proteins of the ECDs (Walsh and Doherty, 1997; Drosopoulos et al., 1999; Wang and Bixby, 1999). To investigate these interactions for cPTPRO, we fused the cDNA encoding the ECD of cPTPRO to the cDNA encoding the Fc domain of mouse IgG-1 (mIgG; Fig. 2, A and B) . This construct was used to express the fusion protein (cPTPRO–Fc) in stably transfected CHO cells, and purified with anti-mIgG agarose. As expected, the purified protein migrated as a 170-kD band under reducing conditions and as a 350-kD dimer under nonreducing conditions (Fig. 2 C). Bands of these same sizes were identified by an anti-cPTPRO antibody on Western blots of the purified proteins (Fig. 2 D), confirming the presence of the cPTPRO ECD in the fusion protein.

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