The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

We have examined the signal transduction pathways of the PDGF alpha‐ and beta‐receptors, in order to characterize the specificity of each receptor type in the signaling. Porcine aortic endothelial cell lines expressing equal levels of either PDGF alpha‐ or beta‐receptors were established. The alpha‐ and beta‐receptor cells responded mitogenically to stimulation with the proper PDGF isoforms. Three aspects of actin reorganization were examined after ligand stimulation: loss of stress fibres, appearance of edge ruffles and formation of circular membrane ruffles. The beta‐receptor cells showed a response to ligand stimulation which included all three features. The alpha‐receptor cells exhibited edge ruffles and loss of stress fibres, but circular ruffles could not be found in several independent alpha‐receptor cell lines. The beta‐receptor cells, but not the alpha‐receptor cells, were able to migrate chemotactically towards a concentration gradient of ligand. The molecular basis for the differences in signalling were explored by comparing the pattern of increased phosphorylation of potential substrates for the alpha‐ and beta‐receptors in [32P]orthophosphate labelled intact cells and using an in vitro kinase assay. Certain of the observed substrates were common for the two receptors, whereas others were specific for either one. We conclude that certain of the known PDGF induced cellular effects are transduced only by the beta‐receptor; the presence of alpha‐receptor‐specific substrates suggests that there are also alpha‐receptor‐specific signals, which have yet to be identified.

The primary structure of the human A-ype receptor for platelet-derived growth factor (PDGF) has been determined. A 6.5-kilobase (kb) transcript was identified through low-stringency hybridization with a probe derived from the B-type PDGF receptor cDNA. The sequence of a cDNA clone corresponding to the 6.5-kb transcript contains an open reading frame that predicts a 1089-amino acid growth factor receptor-like molecule, which displays 44% overall amino acid similariy with the PDGF B-type receptor. The two receptors have a similar domain organization, with five immunoglobulin-like domains extracellularly and an intracellular split protein tyrosine kinase domain.

Two novel sites of autophosphorylation were localized to the C‐terminal tail of the PDGF beta‐receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF‐BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C‐gamma (PLC‐gamma) compared to the wild type PDGF beta‐receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC‐gamma could be detected. These data show that tyrosine phosphorylation of PLC‐gamma is dependent on autophosphorylation of the PDGF beta‐receptor at Tyr1009 and Tyr1021.

The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

ABSTRACT.Purpose: To evaluate surgical performance using intracameral mydriatics (ICM) in phacoemulsification surgery in a series of consecutive cases. Methods: In a series of 198 consecutive procedures 50À200 ml of cyclopentolate 0.1%, phenylephrine 1.5% and lidocaine 1% was given intracamerally for mydriasis and anaesthesia. The previous 198 cases, dilated with topical mydriatics, were studied for comparison. Several pre-intra-and postoperative parameters were registered, and the subjective surgical performance was graded after each procedure. In 41 consecutive cases, the change in pulse and oxygen saturation induced by the ICM injection was registered. Results: No increase in operation time or complication rates was seen with ICM, compared to when standard topical mydriatics were used. The subjective surgical performance was ranked as equally good for both groups. Conclusion: From this clinical evaluation, our impression is that ICM performs well in routine phacoemulsification surgery.

We have investigated the possibility of a protein kinase participating in the signal transduction mechanisms of the interleukin-1 (IL-1) type I receptor (IL-1RI). Our data show that a protein kinase was co-precipitated with the IL-1RI from the two murine T helper cell lines D10N and EL-4. The kinase activity was detected in an in vitro kinase assay performed with the immuno beads in the presence of exogenous substrates. IL-1 treatment of the cells resulted in a rapid activation of this protein kinase in a concentration-dependent manner. Both forms of IL-1, IL-1 alpha and IL-1 beta, induced this kinase activity, whereas the IL-1 receptor antagonist (IL-1ra) was inactive. In excess IL-1ra competitively antagonized IL-1 stimulation. In the in vitro kinase assay the exogenous substrates myelin basic protein and histone H1 were phosphorylated, whereas casein or heat-shock protein HSP27 were not accepted, reflecting a certain selectivity of this protein kinase. The IL-1RI co-precipitable protein kinase showed a serine/threonine specificity and was inhibited by staurosporine, but not by inhibitors specific for protein tyrosine kinase or protein kinase C. These results show that a serine/threonine protein kinase directly interacts with the IL-1RI at the plasma membrane level of T helper cells forming a novel type of IL-1 inducible signaling complex. This protein kinase may resemble the link coupling the plasma membrane IL-1 receptor to cytosolic downstream elements in the IL-1 signaling pathway.