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The receptors for insulin and insulin-like growth factor-I (IGF-I) belong to the family of receptor protein tyrosine kinases [1]. Although a vast body of data supports the concept that insulin stimulates cell growth in vitro and in vivo, the question of whether insulin is physiologically a growth factor remains controversial (for review see [2]). Even more controversial is the question of whether insulin is capable of inducing mitogenic effects through its own receptor, or whether the growth-promoting effects of insulin result from its weak interaction with the IGF-I receptor or occur within insulin/IGF-I receptor hybrids [3,4], or via interphosphorylation of the IGF-I receptor by the insulin receptor tyrosine kinase [5]. The response possibly depends on the cell type and its given supply of insulin and IGF-I receptors as well as the subsets of intracellular signalling molecules that are activated by either receptor. (We use the term IGF-I receptor for simplicity to designate the type 1 IGF receptor which binds both IGF-I and II and probably mediates the mitogenic effects of both growth factors [6].) Diabetologia (1997) Summary Insulin has traditionally been considered as a hormone essential for metabolic regulation, while the insulin-like growth factors (IGF-I and IGF-II) are postulated to be more specifically involved in growth regulation. The conventional wisdom is that they share each other's effects only at high concentrations, due to their weak affinity for the heterologous receptor. We discuss here the evidence that in the proper cellular context, insulin can be mitogenic at physiologic concentrations through its own receptor. We studied the insulin and IGF-I binding characteristics of a new model suitable for analysing insulin receptor mediated mitogenesis; that is, a T-cell lymphoma line that depends on insulin for growth, but is unresponsive to IGFs. The cells showed no specific binding of 125 I-IGF-I and furthermore, no IGF-I receptor mRNA was detected by RNAse protection assay in the LB cells, in contrast with mouse brain and thymus. The cells bound at saturation about 3000 insulin molecules to receptors that had normal characteristics in terms of affinity, kinetics, pH dependence and negative co-operativity. A series of insulin analogues competed for 125 I-insulin binding with relative potencies comparable to those observed in other insulin target cells. The full sequence of the insulin receptor cDNA was determined and found to be identical to the published sequence of the murine insulin receptor cDNA. The LB cell line is therefore an ideal model with which to investigate insulin mitogenic signalling without interference from the IGF-I receptor. Using this model, we have started approaching the molecular basis of insulin-induced mitogenesis, in particular the role of signalling kinetics in choosing between mitogenic and metabolic pathways. [Diabetologia (1997) 40: S 25-S 31]

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlumydomonus reinhurdtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine a-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.In response to a sudden elevation of temperature, a number of specific genes, termed heat-shock genes, are activated in both procaryotic and eucaryotic cells [ 11. Following induction, a rapid transcription and preferential translation of the corresponding heat-shock mRNAs takes place and results in a significant accumulation of the heat-shock proteins [2]. In accordance with the universality of the heat-shock response, a relatively high degree of similarity characterizes heat-shock proteins from various organisms. In higher plants smallmolecular-mass heat-shock proteins (1 5 -30 kDa) represent a particularly prominent and heterogeneous group of proteins coded by multigene families [3 -61. The complexity and the diversity in the sizes of the low-molecular-mass heat-shock proteins seem to indicate a lower degree of similarity, in contrast to the high degree of conservation in the amino acid sequences of the high-molecular-mass heat-shock proteins common to both the plant and animal kingdoms. Like their high-molecular-mass counterparts, the small heat-shock proteins in plants are nuclear-coded and translated on cytosolic ribosomes. The majority of them remain in the cytosol and become associated with the cytoskeleton...

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