Two classes of receptors with distinct affinities for nerve growth factor (NGF) have been identified. The low affinity receptor (Kd approximately 10(-9) to 10(-8) M) is a cysteine-rich glycoprotein encoded by the previously characterized LNGFR gene. The structural nature of the high affinity receptor (Kd approximately 10(-11) to 10(-10) M) has yet to be established. In this study we show that the product of the human trk proto-oncogene (gp140trk) binds NGF with high affinity. Moreover, NGF could be chemically cross-linked to the endogenous gp140trk present in rat PC12 pheochromocytoma cells as well as to gp140trk ectopically expressed in mouse fibroblasts and in insect Sf9 cells. High affinity binding of NGF to gp140trk can occur in the absence of low affinity LNGFR receptors, at least in nonneural cells. Addition of NGF to PC12 cells elicits rapid phosphorylation of gp140trk on tyrosine residues and stimulates its tyrosine kinase activity. These results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.

The trkB gene encodes a tyrosine kinase receptor, gp145trkB, for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). To understand the role of gp145trkB in the nervous system, we have investigated its expression in embryonic rat hippocampal pyramidal cell cultures and examined the effects of BDNF on signal transduction in the primary neurons. The expression of trkB transcripts was established by PCR analysis and in situ hybridization. In addition to gp145trkB, the pyramidal neuronal cultures expressed transcripts specific for the NT-3 receptor gp145trkC, but not for the high-affinity NGF receptor gp140trk or for p75LNGFR, a low-affinity receptor for all known members of the NGF family of neurotrophins including the gp145trkB ligands, BDNF and NT-4. The presence of gp145trkB receptors in the primary neuronal cultures was confirmed by immunocytochemical analysis in which > 90% of the cells stained with affinity-purified polyclonal antibodies to gp145trkB. Immunoblots using this antibody revealed a single approximately 140 kDa protein in both adult hippocampus and pyramidal cultures. Addition of recombinant BDNF to these cultures induced the tyrosine phosphorylation of gp145trkB, as determined by antiphosphotyrosine staining of gp145trkB immunoprecipitates. Moreover, BDNF treatment activated the microtubule-associated protein (MAP) kinases, as determined by an increase in MAP2 phosphorylation in vitro. Both the 41 and 44 kDa forms of MAP kinase were activated by BDNF. BDNF also increased c-fos expression in over 90% of the cells. These results indicate that gp145trkB does not require p75LNGFR to form a functional receptor for BDNF in hippocampal pyramidal neurons.

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Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of β-3-receptor agonists. These include: (i) the microbial reduction of 4-benzyloxy-3-methanesulfonylamino-2′-bromoacetophenone 1 to the corresponding (R)-alcohol 2 by Spingomonas paucimobilis SC 16113. In the biotransformation process, a reaction yield of >85% and an optical purity of 99.5% were obtained for the desired (R)-alcohol 2; (ii) the enzymatic resolution of racemic α-methyl phenylalanine amide, 3, and α-methyl-4-hydroxyphenylalanine amide, 5, by amidase from Mycobacterium neoaurum ATCC 25795 to prepare the corresponding (S)-amino acids 4 and 6. Reaction yields of 49.9 and 49 M% (theoretical maximum yield 50 M%) and optical purities of 99 and 94% were obtained for the desired (S)-amino acids 4 and 6, respectively; (iii) the asymmetric hydrolysis of methyl-(4-methoxyphenyl)-propanedioic acid, ethyl diester, 7, to the corresponding (S)-monoester 8 by pig liver esterase. A reaction yield of 96 M% and an optical purity of 96% were obtained for (S)-monoester 8 when reactions were carried out in a biphasic system containing 10% ethanol at 10°C.Much attention is currently focused on the interaction of small molecules with biological macromolecules. The search for selective enzyme inhibitors and receptor agonists or antagonists is one of the keys for target-oriented research in the pharmaceutical industry. Increasing understanding of the mechanism of drug interaction on a molecular level has led to growing awareness of the importance of chirality as the key to the efficacy of many drug products. It is now known that in many cases only one stereoisomer of a drug substance is required for efficacy; the other stereoisomer is either inactive or exhibits considerably reduced acitivity. Pharmaceutical companies are aware that, where the switch from racemate drug substance to enantiomerically pure compound is feasible, new drugs should be homochiral to avoid the possibility of side effects caused by an undesirable stereoisomer. There is the opportunity to double the use of an industrial process by obtaining a separate patent on the use of a single stereoisomer as a more efficient drug. The physical advantages of enantiomers over racemates may confer processing and formulation advantages, as well as lower doses.The advantages of microbial or enzyme-catalyzed reactions over chemical reactions are that they are stereoselective and can be carried out at ambient temperature and atmospheric pressure. These minimize problems of isomerization, racemization, epimerization, and rearrangement, which generally occur during chemical processes. Biocatalytic processes are usually carried out in aqueous solution. This avoids the use of solvent and other environmentally harmful chemicals used in the chemical processes. Furthermore, microbial cells or enzymes derived from biocatalysis can be immobilized and reused for many cycles. Recently, a number of review articles (1-9) have been published on the use of enzymes in organic synthesis.β-Adrenocepto...