Ligand stimulation of the platelet-derived growth factor (PDGF) P-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF ,8-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF ,-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFRjY716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF ,8-receptors. The mutant PDGFRjIY716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF j-receptor.Platelet-derived growth factor (PDGF) is a connective tissue cell mitogen, consisting of dimers of disulfide-bonded A and B polypeptide chains, which combine to form the three isoforms, PDGF-AA, -AB, and -BB (for a review, see reference 21). Two types of PDGF tyrosine kinase receptors have been identified and cloned. The ox-receptor binds both A and B chains with high affinity, whereas the p-receptor binds only the B chain.Signal transduction through tyrosine kinase receptors follows a general scheme whereby ligand binding induces dimerization or oligomerization of receptor molecules, activation of the receptor tyrosine kinase, and autophosphorylation in trans on multiple tyrosine residues in the intracellular region (reviewed in reference 44). Thereby, binding sites are created for intracellular signal transduction molecules, containing one or two copies of Src homology 2 (SH2) domains. There are two principal groups of signal transduction molecules: those which are equipped with a catalytic domain, and those which lack such domains but which serve as adaptors and associate with catalytically active molecules (50). For the PDGF P-receptor, considerable information has accumulated regarding the positions of autophosphorylation sites as well as which signal transduction molecules interact with individual sites. Thus, members of the Src family, i.e., phospholipase C--y, the Ras GTPase-activating protein (GAP), phosphatidylinositol 3'-kinase (P13-kinase), the phosphotyrosine phosphatase PTP1D/ Syp, and the adaptor molecules Shc in the PDGF P-receptor (reviewed in reference 9). The specificity of these in...

The platelet-derived growth factor (PDGF) a and p receptors undergo dimerization as a consequence of ligand binding. Depending on the PDGF isoform (PDGF-AA, -AB or -BB), homodimers or heterodimers of receptors are formed. In this study, we have used transfected porcine aortic endothelial cells, coexpressing cDNAs for the a receptor and the p receptor at comparable levels, to investigate the properties of the up-heterodimeric receptor complex. PDGF-AB, which mainly induced ap-heterodimeric complexes, was the most efficient isoform for stimulating mitogenicity. Actin reorganization, in the form of circular membrane ruffling and chemotaxis, was induced by PDGF-AB and PDGF-BB, but not by PDGF-AA, thus indicating that the p receptor in the homodimeric or heterodimeric configuration was required for induction of motility responses. The molecular basis for the apparent receptor dimer-specific properties was examined by analyzing receptor autophosphorylation and phosphorylation of substrates. The a receptor was found to be phosphorylated at an additional tyrosine residue, Tyr754, in the heterodimeric complex as compared to the aa receptor homodimer. Phosphorylation of this tyrosine residue could permit the binding of a specific signal-tranducing protein. A candidate is a 134000-Mr protein, which was shown to associate preferentially with the a receptor in the heterodimeric receptor complex. It is possible that phosphorylated Tyr754 in the a receptor mediates activation of specific signal-tranducing molecules like the 134 000-M, substrate, and thereby initiates signal-tranduction pathways from the ap receptor heterodimer, which are distinct from those initiated via homodimeric receptor complexes.

Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC-gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma.

The human retinoic acid receptor u was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA binding activities in gel-retardation assays. Ligand binding properties were measured in a charcoal absorption assay. The dissociation constant for highly specific bound retinoic acid was found to be 0.67 nM. The affinity of several synthetic retinoids to thc recombinant protein was determined and compared to their biological activity. Some of the values presented here differ significantly from those published earlier for the receptor or its isolated hormonebinding domain.The nuclear receptors for retinoids play a major role in the regulation of genes involved in cell differentiation and growth. Their natural ligand, the vitamin A derivative alltrans-retinoic acid, has been reported to be a possible morphogen in the embryonic tissues of frogs and chicken.

We have screened the sequence of the 394 base pairs upstream of the main transcriptional start site of the promoter of the human parathyroid hormone (PTH) gene for well-known protein recognition motifs with the aim to identify potential positive or negative regulatory elements. Within this region we found a potential cAMP-response element (CRE) besides several other putative binding sites for transcription factors. We fused promoter regions that contain this element and extend beyond the transcription start site to an appropriate reporter gene (CAT) and transfected different cell lines with these constructs. Transient expression of the CAT gene from these hybrid genes could be shown to be significantly stimulated by forskolin or isoproterenol thus proving the responsiveness of the whole promoter region towards elevated cAMP levels. DNase I protection studies revealed protein binding around the putative CRE (PTH-CRE) and an adjacent CCAAT element. Gel retardation assays with the PTH-CRE as well as the well-characterized CRE from the rat somatostatin promoter indicated specific binding of the same protein to both elements, although with a slightly reduced affinity of the PTH-CRE. Both of these elements were also able to confer cAMP-responsiveness to a heterologous promoter.

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

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