Affinity labeling and partial purification of nerve growth factor receptors from rat pheochromocytoma and human melanoma cells.

Abstract

A cell surface glycoprotein receptor for nerve growth factor (NGF) has been identified by covalent crosslinking to 125I-labeled NGF (125I-NGF). Either ethyldimethylisopropyl-aminocarbodiimide or hydroxysuccinimidyl-p-azidobenzoate causes highly specific crosslinking of 125I-NGF to a similar receptor species on rat pheochromocytoma PC12 cells and on human melanoma A875 cells. The NGF-receptor complex migrates as a broad band in NaDodSO4/polyacrylamide gel electrophoresis with an apparent Mr of approximately equal to 100,000. Because the NaDodSO4-denatured complex apparently contains a single Mr 13,000 NGF chain, the apparent molecular weight of the receptor itself is 87,000. Inhibition of protein glycosylation by tunicamycin generates smaller 125I-NGF-receptor complexes. The mobility of the smallest of these in NaDodSO4 gel electrophoresis corresponds to a Mr of 90,000 for the complex and, hence, 77,000 for the carbohydrate-deficient receptor. A second NGF-receptor complex with a Mr of 225,000 also is obtained from A875 cells but only occasionally from PC12 cells. Tunicamycin treatment decreases the molecular weight of these species by 10,000-15,000. Substantial purification of the Mr 100,000 NGF-receptor complex was achieved by lectin affinity chromatography on columns of wheat germ agglutinin linked to Affi-Gel 15. The specific absorption of NGF-receptor complexes to these columns indicates that the receptor is a glycoprotein that contains N-acetyl-D-glucosamine.

• Authors: P M Grob, C H Berlot, M A Bothwell
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 80 Issue 22 Pg. 6819-23 (Nov 1983) ISSN: 0027-8424 [Print] United States
• PMID: 6316327 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Affinity Labels
• Cross-Linking Reagents
• Nerve Growth Factors
• Receptors, Cell Surface
• Receptors, Nerve Growth Factor

Topics

• Adrenal Gland Neoplasms (metabolism)
• Affinity Labels (pharmacology)
• Animals
• Cell Line
• Cross-Linking Reagents (pharmacology)
• Humans
• Melanoma (metabolism)
• Molecular Weight
• Nerve Growth Factors (metabolism)
• Pheochromocytoma (metabolism)
• Rats
• Receptors, Cell Surface (drug effects, isolation & purification, metabolism)
• Receptors, Nerve Growth Factor