Nerve growth factor binding domain of the nerve growth factor receptor.

Abstract

A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptors to bind NGF was assayed.In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extraceflular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terinal deletions, in which either the first cysteine-rich sequence or the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteinerich sequences of the NGF receptor contain the NGF binding domain.The role of nerve growth factor (NGF) in the development and survival of peripheral sensory and sympathetic neurons has been well characterized (1). Studies have also suggested a role for NGF in the development and maintenance of cholinergic neurons in the mammalian forebrain (2).The first step in the mechanism of action of NGF is the interaction of NGF with its specific receptor [the NGF receptor (NGFR)] on responsive cells or cell lines. There are two types or states of the NGFR as determined by binding studies (3). On sensory and sympathetic neurons, these receptors are described as high-affinity (Kd = 10 pM) and low-affinity (Kd = 1 nM) NGFRs, respectively. Although the NGFRs in PC12 cells differ less in their Kd values, they share many features of the receptors on primary neurons, including a slower rate ofdissociation of NGF from the high-compared to the low-affinity receptor (4). The biological response of NGF in neuronal cells correlates with occupancy of the high-afflinity receptor. Indirect evidence suggests that the high-affinity NGFR comprises the low-affinity receptor complexed with an additional cytoplasmic or membraneassociated protein or proteins (5,6). The low-affinity receptor has been cloned in rat (7), chicken (8), and human (9), and the three receptors share considerable homology.Recent studies have shown that Schwann cells in rat sciatic nerve express high levels of the low-affinity NGFR after nerve crush or transection (10, 11). This expression is repressed once contact is reestablished between the Schwann cells and the regenerating axons. These low-affinity receptors may bind the NGF that is synthesized by nonneuronal cells distal to a peripheral nerve injury and create a localized source of NGF on the surface of the Schwann cells over which the advancing neuronal growth cone migrates (12).In addition, a truncated form of the low-affinity receptor is secreted into the extracellular medium of cultured Schwann cells, PC12 cells, and super...

Bombesin/GRP Stimulates a Protein Kinase in Swiss 3T3 Cells That Phosphorylates Microtubule-Associated Protein Kinase.

“…Recently, a cDNA for the bombesin/GRP receptor was cloned, and the protein has been shown to have a transmembrane topology similar to that of other G protein-coupled receptors [2,3]. There is some evidence that cellular responses to bombesin/GRP may be regulated by G-proteins coupled to the GRP receptor in a manner analogous to the coupling of adenylate cyclase to receptors via G-proteins [24][25][26][27].…”

“…Key Words: bombesin/GRP, MAP kinase, Swiss 3T3, nerve growth factor, epidermal growth factor Bombesin/gastrin-releasing peptide (GRP) and epidermal growth factor (EGF) receptors exert their mitogenic effects in Swiss 3T3 cells by distinct pathways [1]. A cDNA for the bombesin/GRP receptor has been cloned [2,3], and the protein has been shown to have a transmembrane topology like that of other G protein-coupled receptors. Although tyrosine kinase activity has been associated with the receptor function as reported by Isacke et al .…”

Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae

“…The 48 and 36 kDa products were recognized in Western analysis by antihspl50A-NGFRe antiserum, but neither of them by antihspl50 antiserum, whereas the intact fusion protein was recognized by both (not shown). The 48 kDa form most probably was the correct product, NGFR~, because it had the expected size [15]. Moreover, NGFR~ expressed in the absence of the hspl50A-carrier by the aid of the invertase signal peptide also migrated like a 48 kDa protein (see below).…”

Cloning and Characterization of a Specific Receptor for Mouse Oncostatin M

“…The amplified cDNA was subcloned into the expression vector pBJ5 (21). To transiently express mouse OSM, 5 g of PBJ5-OSM plasmid was transfected as a calcium phosphate precipitate into 10 6 293T cells that had been plated on fibronectin-treated plates (2).…”