A.M. de Vos

Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression.

Glucose homeostasis is controlled by a glucose sensor in pancreatic beta-cells. Studies on rodent beta-cells have suggested a role for GLUT2 and glucokinase in this control function and in mechanisms leading to diabetes. Little direct evidence exists so far to implicate these two proteins in glucose recognition by human beta-cells. The present in vitro study investigates the role of glucose transport and phosphorylation in beta-cell preparations from nondiabetic human pancreata. Human beta-cells differ from rodent beta-cells in glucose transporter gene expression (predominantly GLUT1 instead of GLUT2), explaining their low Km (3 mmol/liter) and low VMAX (3 mmol/min per liter) for 3-O-methyl glucose transport. The 100-fold lower GLUT2 abundance in human versus rat beta-cells is associated with a 10-fold slower uptake of alloxan, explaining their resistance to this rodent diabetogenic agent. Human and rat beta-cells exhibit comparable glucokinase expression with similar flux-generating influence on total glucose utilization. These data underline the importance of glucokinase but not of GLUT2 in the glucose sensor of human beta-cells.

Nerve growth factor: structure and function.

Abstract. Neurotrophins are critical for the development and maintenance of the peripheral and central nervous system. These highly homologous, homodimeric growth factors control cell survival, differentiation, growth cessation, and apoptosis of sensory neurons. The biological functions of the neurotrophins are mediated through two classes of cell surface receptors, the Trk receptors and the p75 neurotrophin receptor (p75NTR). Nerve growth factor (NGF), the best characterized member of the neurotrophin family, sends its survival signals through activation of TrkA and can induce cell death by binding to p75NTR. Recent domain deletion and mutagenesis studies have identified the membrane-proximal domain of the Trks as necessary and sufficient for ligand binding. Crystal structures of this domain of TrkA, TrkB, and TrkC, and an alanine scanning analysis of this domain of TrkA and TrkC have allowed identification of the ligand- binding site. The recent crystal structure of the complex between NGF and the ligand-binding domain of TrkA defines the orientation of NGF in the signaling complex, and eludicates the structural basis for binding and specificity in the family. Further structural work on NGF-TrkA- p7SNTR complexes will be necessary to address the many remaining questions in this complex signaling system.

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta‐cells differ in their individual rates of glucose‐induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta‐cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta‐cells were 2‐ to 4‐fold higher than in low responsive beta‐cells, whereas their leucine and glutamine oxidation was only 10–50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13–19 pmol/min/10(3) beta‐cells) were at least 50‐fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3–0.7 pmol/min/10(3) beta‐cells) ranged within those of total glucose utilization (0.2–0.4 pmol/min/10(3) beta‐cells). High responsive beta‐cells exhibited a 60% higher glucokinase activity than low responsive beta‐cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta‐cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta‐cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.

Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor.

The structure of the ternary complex between ovine placental lactogen (oPL) and the extracellular domain (ECD) of the rat prolactin receptor (rPRLR) reveals that two rPRLR ECDs bind to opposite sides of oPL with pseudo two-fold symmetry. The two oPL receptor binding sites differ significantly in their topography and electrostatic character. These binding interfaces also involve different hydrogen bonding and hydrophobic packing patterns compared to the structurally related human growth hormone (hGH)–receptor complexes. Additionally, the receptor–receptor interactions are different from those of the hGH–receptor complex. The conformational adaptability of prolactin and growth hormone receptors is evidenced by the changes in local conformations of the receptor binding loops and more global changes induced by shifts in the angular relationships between the N- and C-terminal domains, which allow the receptor to bind to the two topographically distinct sites of oPL.

CRYSTALLIZATION OF OVINE PLACENTAL LACTOGEN IN A 1:2 COMPLEX WITH THE EXTRACELLULAR DOMAIN OF THE RAT PROLACTIN RECEPTOR

Growth hormone and prolactin control somato-lactogenic biology. While high-resolution crystal structures have been determined for receptor complexes of human growth hormone, no such information exists for prolactin. A stable 1:2 complex was formed between ovine placental lactogen, a close prolactin homologue, and two copies of the extracellular portion of the rat prolactin receptor. Using synchrotron radiation, native data have been collected to 2.3 A. Crystals contain one complex per asymmetric unit. The crystal structure of this complex will shed light on the structural reasons for cross-reactivity and specificity among the endocrine hormones, placental lactogen, prolactin and growth hormone.

From big molecules to smaller ones

Protein-protein interfaces are considerably larger than small molecule-protein interfaces. Nonetheless, mutational studies show only a small portion of the interface between hGH and its receptor is crucial for tight binding. This is likely a consequence of the fact that only a small number of contacts are necessary to generate high affinity. The interface is capable of adapting to mutational changes by reorganizing contact side chains. This plasticity affords the protein interface many options to adapt to mutational changes in its binding partner as they co-evolve.

Three-Dimensional Structure of ras p21 Proteins

ras genes (review in Barbacid, 1987) have been found in a large number of eukaryotic organisms, from Saccharomyces and Drosophila to chicken, rat, and man. Moreover, ras gene products of various species show a very high degree of homology: even between proteins from yeast and man there is approximately 54% identity between corresponding amino acids, and ras proteins from chicken and man differ only in three amino acids. Such evolutionary conservation implies an important cellular function for these proteins, and they have indeed been implicated in playing a crucial role in cell proliferation and terminal differentiation. Based on these observations and on biochemical similarities with G-proteins, it is thought that ras proteins participate as signal transducers at the beginning of the cascade of reactions leading to various essential cellular processes.