Expression of Grb7 growth factor receptor signaling protein in kidney development and in adult kidney.

Grb7, a signaling protein whose physiological function is unknown, binds receptor tyrosine kinases important for normal kidney development. By investigating and correlating Grb7 gene expression with that reported for Grb7-binding receptors, we provide clues to Grb7 function(s). RT-PCR and immunoblot were used to demonstrate Grb7 gene and protein expression in the mature kidney. Additional RT-PCR studies detected gene expression in all microdissected adult nephron segments examined, except glomeruli, and in the mouse metanephric kidney from embryonic day 11 (E11) through to day 17 (E17). In situ hybridization at E14 demonstrated the following cellular pattern of localization: Grb7 mRNA in metanephric epithelia of mesenchymal and ureteric bud origin; no expression in the undifferentiated mesenchyme; and little expression in podocyte-destined cells or primitive glomeruli. Grb7 mRNA was also present in the epithelia of the lung and gut at E14. Thus Grb7 may have a basic function in growth factor signaling in terminally differentiated epithelia along the nephron and in developing epithelia in the kidney, lung, and gut. It is localized in a pattern permissive for a role in Her2 and Ret receptor signaling.

Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor.

src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.

Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase C gamma 1.

The effects of platelet-derived growth factor (PDGF) on cytosolic free Ca2+ concentration ([Ca2+]i) and inositol phosphates were studied in NIH-3T3 fibroblasts transfected with cDNA for phospholipase C gamma 1 (PLC gamma 1) to yield a 7-fold overexpression of this enzyme, compared with cells containing normal levels of PLC gamma 1. In a study published recently [Margolis, Zilberstein, Franks, Felder, Kremer, Ullrich, Rhee, Skorecki & Schlessinger (1990) Science 248, 607-610] it was reported that this overexpression of PLC gamma 1 caused a specific potentiation of the inositol phosphate response to PDGF, but this was not associated with an enhancement of the [Ca2+]i response. In the present study, measurements of the time course and isomeric profile of PDGF-induced inositol phosphate formation demonstrated that the initial rate of Ins(1,4,5)P3 formation was also enhanced in the PLC gamma 1-overexpressing cells, yielding a 10-fold greater increase at 1 min compared with the parental NIH-3T3 cells. By contrast, bradykinin-induced phosphoinositide metabolism was unchanged in PLC gamma 1-transfected cells. Measurements of [Ca2+]i in cell populations and single cells showed a significant latent period following PDGF addition prior to the [Ca2+]i increases in both cell lines, which decreased in a dose-dependent manner with increasing PDGF concentration. The duration of the latent period was decreased and the maximal rate of [Ca2+]i rise was increased in the PLC gamma 1-overexpressing cells at all doses of PDGF examined. In single-cell measurements these cells also responded to PDGF with a greater peak amplitude of [Ca2+]i. Both intracellular Ca2+ mobilization and Ca2+ influx across the plasma membrane were enhanced in the PLC gamma 1-overexpressing cells. There was no difference between the two cell lines in either the latency or the magnitude of the [Ca2+]i increases induced by bradykinin. These data provide further evidence that PLC gamma 1 is responsible for the PDGF-induced stimulation of Ins(1,4,5)P3 formation. Moreover, in contrast to earlier conclusions, the modified kinetics of the [Ca2+]i changes in PLC gamma 1-overexpressing cells suggest that Ins(1,4,5)P3 does play a predominant second messenger role in the PDGF-induced [Ca2+]i increases. The data also indicate that the latent period may be a function of the time required to reach a threshold level of Ins(1,4,5)P3, rather than an intrinsic property of the PDGF receptor.

Distinct structural specificities for functional coupling of the epidermal growth factor receptor to calcium-signalling versus phospholipase A2 responses.

Activation of phospholipase C (PLC), leading to a rise in cytosolic Ca2+, and of phospholipase A2 (PLA2) leading to a release of arachidonic acid, are among the early transmembrane signalling events that have been demonstrated in response to occupancy of the epidermal growth factor (EGF) receptor. The tyrosine kinase activity of the receptor has been shown to be necessary for both of these responses. This requirement for the tyrosine kinase activity could conceivably implicate a role for receptor autophosphorylation in the activation of PLA2. We now demonstrate that coupling of the EGF receptor to PLA2 was not impaired in a deletion mutant (CD126) devoid of the 126 amino acids from the C-terminus which include four major autophosphorylation sites. Functional coupling of the EGF receptor to PLA2 was demonstrated using three different experimental designs: (1) release of [14C]arachidonic acid from prelabelled intact cells. (2) release of [3H]arachidonic acid from prelabelled cells permeabilized with glass beads, and (3) direct measurement of PLA2 enzymic activity in cell-free extracts using an 'in vitro' assay employing exogenous phospholipid substrate. Functional coupling of the EGF receptor to PLA2 occurred despite the absence of a demonstrable Ca(2+)-signalling response and the detection of diminished but persistent PLC-gamma phosphorylation on tyrosine residues in the CD126 deletion mutants. These results point to a clear distinction in the biochemical mechanism and role for receptor autophosphorylation in functional coupling of the EGF receptor to PLA2 activation versus Ca2+ signalling.

The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.

We have previously reported that epidermal growth factor (EGF) activates phospholipase A2 (PLA2) independently of phospholipase C (PLC) in renal mesangial cells. In this study we use NIH 3T3 cell lines transfected with the normal EGF receptor (HER14 cells) or with EGF receptor defective in tyrosine kinase activity (K721A cells), to determine whether the intrinsic tyrosine kinase activity of the EGF receptor is required for the PLC-independent activation of PLA2. Intact cells were preincubated with EGF or other ligands, and then PLA2 activity was assayed in cell-free extracts with 1-stearoyl-2-[14C]arachidonyl phosphatidylcholine as the substrate. In HER14 cells, EGF increased PLA2 activity by 226 +/- 30%, and the tumour promoter phorbol myristate acetate (PMA) increased activity by 223 +/- 30%. The effect of EGF was not mediated through protein kinase C (PKC), whose activation by EGF requires tyrosine kinase activity, since raising intracellular Ca2+ alone with the Ca2+ ionophore A23187 did not mimic its effect, and the effect of EGF persisted in PKC-down-regulated cells. In K721A cells EGF was ineffective, whereas PMA was still active. Furthermore, in intact HER14 cells prelabelled with [14C]arachidonate, EGF-stimulated release of [14C]arachidonic acid was synergistic with A23187, but was unaccompanied by a rise in [14C]diacylglycerol. EGF had no effect on [14C]arachidonic acid release in intact K721A cells. We conclude that the tyrosine kinase activity of the EGF receptor is necessary for the PLC-independent stimulation of PLA2 by EGF.

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor.

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.

Epidermal growth factor stimulates phospholipase A2 in vasopressin-treated rat glomerular mesangial cells.

Epidermal growth factor (EGF) enhances vasopressin- and ionophore-A23187-induced prostaglandin production and arachidonate release by rat glomerular mesangial cells in culture. The purpose of the present study was to delineate the phospholipid pathways involved in this effect. In cells labelled with [14C]arachidonate, EGF significantly enhanced the free arachidonate released in response to A23187 or vasopressin without enhancing the production of [14C]arachidonate-labelled diacylglycerol. EGF increased the [14C]arachidonate-labelled phosphatidic acid formed in response to vasopressin, but to a much smaller extent than it increased free arachidonate release. These results indicate that activation of phospholipase C is not sufficient to explain the increase in free arachidonate release observed on addition of EGF. To examine if EGF enhanced phospholipase A2 activity, mesangial cells were labelled with [2-2H]glycerol and [14C]-arachidonate, and the formation of arachidonate-poor lysophospholipids was studied. When combined with vasopressin, EGF significantly enhanced the formation of arachidonate-poor lysophospholipids as compared with vasopressin alone. The fate of exogenously added lysophosphatidylcholine was not altered after stimulation with vasopressin plus EGF, indicating that decreased deacylation or reacylation of the lysophospholipids was not responsible for their accumulation. Taken together, these results indicate that EGF enhances free arachidonate release by activation of phospholipase A2. The signalling mechanism responsible for the change in phospholipase A2 activity is not known, but could conceivably involve phosphorylation of modulating proteins such as lipocortin or G-proteins.

The urine osmolal gap: a clue to estimate urine ammonium in "hybrid" types of metabolic acidosis.

The urine osmolal gap is defined as the difference between measured urine osmolality and the sum of the concentrations of sodium, potassium, chloride, bicarbonate, urea and glucose. Normally, this gap is 80-100 mosmol/kg H2O. A determination of the urine osmolal gap may be useful to ascertain the etiology of metabolic acidosis which is of the mixed wide and normal plasma anion gap type ("hybrid" metabolic acidosis). For example, with "hybrid" metabolic acidosis, a low urine osmolal gap will suggest the absence of excessive organic aciduria (ketoacidosis) and the basis of the normal anion gap type of acidosis will be determined by the urine anion gap or "net charge". Where "hybrid" metabolic acidosis has occurred due to wide anion gap metabolic acidosis with loss of organic acid anion in the urine, the urine osmolal gap will be high and can be used in a semi-quantitative fashion to estimate the sum of urinary ammonium plus ketone body anion concentrations.

Epidermal growth factor is synergistic with phorbol esters and vasopressin in stimulating arachidonate release and prostaglandin production in renal glomerular mesangial cells.

Epidermal growth factor (EGF) is produced in large quantities by the kidney. We identified EGF-binding sites on cultured rat renal glomerular mesangial cells. These cells serve as a model system for the investigation of renal prostaglandin biosynthesis. Since EGF has been shown to modulate phospholipase activity in other cell lines, we studied the ability of EGF to increase arachidonate release and prostaglandin E2 (PGE2) production in mesangial cells. We found that EGF stimulated arachidonate release and PGE2 production in the presence of the Ca2+ ionophore A23187. This stimulation was markedly potentiated by the addition of phorbol myristate acetate (PMA), which activates protein kinase C. However, down-regulation of protein kinase C by prolonged PMA treatment did not block the ability of EGF to stimulate PGE2 production in the presence of A23187. EGF also markedly potentiated the stimulation of PGE2 production by vasopressin, which increases intracellular Ca2+ and activates protein kinase C in these cells. The stimulatory effects of EGF were not the result of prolongation or enhancement of an increase in intracellular Ca2+ produced by ionophore or vasopressin. Furthermore, the synergistic interaction of EGF with PMA and vasopressin occurred despite the fact that these agents markedly decreased EGF binding in mesangial cells, presumably owing to protein-kinase-C-mediated phosphorylation of the EGF receptor. We conclude that there exists a distinct pathway for EGF-stimulated arachidonate release and PGE2 production in rat renal glomerular mesangial cells, which is synergistic with, but not dependent on, activation of protein kinase C. In contrast with long-term mitogenic responses to EGF, this rapid response may allow delineation of the membrane phospholipid changes and signalling steps involved in this aspect of EGF action.

The Spitzer-Weinstein syndrome: one form of type IV renal tubular acidosis and its response to hydrochlorothiazide.

A twelve-year-old girl with persistent hyperkalemia, metabolic acidosis, normal blood pressure and glomerular filtration rate, and short stature (first percentile for height) was studied using metabolic balance techniques. Prior to therapy with hydrochlorothiazide (HCTZ), urinary potassium and acid excretion were low and urine pH was inappropriately high at 5.8. HCTZ (25 mg orally per day) (1 mg/kg) was then started and rapidly corrected her serum electrolytes. The therapy with HCTZ was associated with a diuresis, a decrease in urine pH to 4.8, and concomitant increases in potassium, titratable acid (TA) and ammonium excretion. The increase in TA excretion was explicable, in part, to the decrease in urine pH and, in part, to the considerable increase in phosphate excretion (from 56 to 81 mmol/d). Plasma renin activity and plasma aldosterone increased markedly following HCTZ but urinary prostaglandin E (PGE) excretion was unchanged. These observations suggest that administration of HCTZ in this setting increases hydrogen ion secretion. It is unclear whether this effect is a direct consequence of HCTZ at the level of the tubule or is secondary to some other action of HCTZ. However, it is clear that this effect is not related to an alteration in PGE excretion.

Ammonia production and amino acid metabolism by rat renal papillary epithelial cells in culture.

A significant percentage of excreted ammonium is added to tubular fluid along the medullary collecting duct. However, it is not clear whether this ammonia is produced in the cortex and delivered into the medulla or is produced directly by medullary cells. To address this issue, rat epithelial cells derived from the renal papilla were grown in continuous culture and their ability to generate ammonia was examined. When grown in Dulbecco's modified Eagle's medium with 4 mM glutamine, these cells produced ammonia at a rate of approximately 27 nmol/10(6) cells/h. When these cells were grown in minimum essential medium without glutamine, ammonia production fell to 7 nmol/10(6) cells/ h. Increasing the glutamine concentrations of minimum essential medium to 4 mM increased ammonia production to slightly greater than 30 nmol/10(6) cells/ h. Increasing the media concentration of glutamate, glycine, or asparagine resulted in no significant increase in ammoniagenesis. Analysis of media amino acid concentration revealed that glutamine was the main amino acid consumed while alanine was the predominant amino acid produced. The glutaminase activity of these cells appears to be primarily phosphate-dependent, similar to that observed in vitro in papillary tubules. Alterations of K+ or H+ ion concentration did not alter ammoniagenesis, but addition of 2.5 mM ammonium chloride significantly reduced net ammonia production. It is concluded that rat papillary epithelial cells have the intrinsic ability to utilize glutamine to generate ammonia and alanine. In vivo ammonia produced locally in the medulla may contribute to final urinary ammonium excretion.