Carboxyl-terminal amino acid residues in elongation factor G essential for ribosome association and translocation.

The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.     

Epidermal growth factor receptor kinase translocation and activation in vivo

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.     

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.     

180-kD ribosome receptor is essential for both ribosome binding and protein translocation

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.     

An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation

The elongation cycle of protein synthesis is completed by translocation, a rearrangement during which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by elongation factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suggests that GTP hydrolysis drives a conformational rearrangement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the energy of GTP hydrolysis is utilized to promote the ribosome rearrangement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA.     

Ultrastructural evidence of oestradiol receptor by immunochemistry

Antiserum against calf uterus oestradiol receptor has been used for detecting oestradiol receptor in rat pituitary cells at the ultrastructural level after immunochemical reaction according to Sternberger. The gonadotropic, lactotropic and somatotropic cells were positive, but not the thyrotropic and corticotropic cells. In peripubertal and adult rats, both cytoplasmic and nuclear receptors were seen, but in a long-term castrated rat, the receptor was found only in the cytoplasm. After oestradiol administration to 21-day-old animals, the cytoplasmic receptor decreased and the nuclear receptor increased in gonadotropic cells, supporting the concept of hormone-receptor complex translocation. Antibodies against α1-foetoprotein demonstrated the presence of this oestrogen-binding plasma protein in all pituitary cells, but only in the cytoplasmic area. These results and the immunological controls related to antibody specificity give the first evidence of steroid receptor at the ultrastructural level.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

The amino-terminal region of toll-like receptor 4 is essential for binding to MD-2 and receptor translocation to the cell surface

Toll-like receptor 4 (TLR4) and MD-2 are pivotal components that elicit inflammatory responses to lipopolysaccharide (LPS). They have been shown to form a physical complex on the cell surface that responds directly to LPS. However, the functional region of TLR4 required for association with MD-2 and LPS responsiveness is poorly understood. To identify the region of TLR4, we created a series of mutants with deletions in the extracellular domain and examined their activities in human embryonic kidney 293 cells. A mutant with a 317-amino acid deletion from the membrane proximal region of TLR4 was capable of associating with MD-2, while only a 9-amino acid truncation of the N terminus severely impaired the interaction. The association between the two molecules was well correlated with TLR4 maturation into an endoglycosidase H-resistant form and the cell surface expression. Mouse MD-2 bound to human TLR4, but its activity to facilitate the cell surface expression of TLR4 and confer LPS responsiveness was much weaker than that of human MD-2, indicating species specificity. A chimeric receptor composed of the N-terminal region of human TLR4 and the adjacent region of mouse TLR4 showed preference for human MD-2 in its transport to the cell surface and responsiveness to LPS. Taken together, the N-terminal region of TLR4 is essential for association with MD-2, which is required for the cell surface expression and hence the responsiveness to LPS.     

Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.     

An essential role for FGF receptor signaling in lens development

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating fundamental processes of induction and differentiation. More recently, studies in the lens have contributed significantly to our understanding of cell cycle regulation and apoptosis. Over 20 years of accumulated evidence using several different vertebrate species has suggested that fibroblast growth factors (FGFs) and/or fibroblast growth factor receptors (FGFRs) play a key role in lens development. FGFR signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration. Here we will review and discuss historical and recent evidence suggesting that (FGFR) signaling plays a vital and universal role in multiple aspects of lens development.     

The unoccupied nuclear oestradiol receptor in the rat uterus and hypothalamus during the oestrous cycle.

The nuclear oestrogen receptor population in the rat uterus contained an unoccupied receptor component that bound oestradiol with the high affinity (Kd congruent to 0.5 nM) characteristic of oestrogen receptors. This unoccupied receptor was present at all phases of the oestrous cycle. Its content changed in parallel with that of the total nuclear receptor during the cycle. Oestradiol administration to the immature rat resulted in increases in the uterine content of long-term nuclear receptors (i.e., those still present 8 h after administration); these increases were due to occupied oestrogen receptors, since the content of unoccupied receptor was unchanged. Our previous experiments [White & Lim (1980) Biochem. J. 190, 833-837] have shown in contrast, that oestradiol administration results in an increase in the content of unoccupied nuclear receptor in the hypothalamus. However, as in the uterus, similar cyclic changes in the content of unoccupied nuclear receptor occurred in parallel with those of the total nuclear receptor population in the hypothalamus. Differences and similarities between the unoccupied nuclear receptor of the uterus and hypothalamus are briefly discussed.     

Functionally essential cytoplasmic domain of the erythropoietin receptor.

The cytoplasmic domains of the erythropoietin receptor essential for signal transduction were identified by assessing a series of truncated and deletional mutant receptors. A 91-amino acid region proximal to the transmembrane domain was required for growth signaling. In this region, residues between 353Pro and 362His and between 278Gln and 308Leu appeared to constitute the essential cytoplasmic domains. These two domains contain the conserved amino acids common in the cytokine receptor superfamily, which indicates that these domains in the cytoplasmic regions of the erythropoietin receptor may be important for interaction with common signal transducers or protein tyrosine kinases.