Biomedical subjects

J I Paul

Publications and source records attributed to J I Paul.

5 recordsLinked to original sources

PubMed1990

Effects of amino acid replacements within the tetrabasic cleavage site on the processing of the human insulin receptor precursor expressed in Chinese hamster ovary cells.

We have studied the specificity requirements for processing of the human insulin proreceptor by successively replacing each basic amino acid in the tetrabasic cleavage site with alanine. These mutated receptor cDNAs have then been overexpressed in Chinese hamster ovary cells, using vectors containing the mouse dihydrofolate reductase gene to amplify the transfected cDNAs in the presence of increasing concentrations of methotrexate. High levels of expression, ranging up to 6 x 10(7) receptors/cell were achieved in these experiments. Replacement of the P1 arginine with alanine led to the complete suppression of processing, as occurs also in a naturally occurring serine mutation at this site (Yoshimasa, Y., Seino, S., Whittaker, J., Kakehi, T., Kosaki, A., Kuzuya, H., Imura, H., Bell, G. I., and Steiner, D. F. (1988) Science 240, 783-787). A small amount of cleavage at alternative sites was detected. Replacement of the P2 arginine or P3 lysine with alanine did not in either case affect conversion to mature alpha and beta subunits, while replacement of the P4 arginine significantly inhibited processing. The binding isotherms for the processed versions of the receptor were comparable to previously published normal values. The unprocessed proreceptor bound insulin normally but was autophosphorylated less efficiently than processed versions of the receptor expressed in the same cells. These results suggest that a single processing protease with trypsin-like specificity may be involved in processing both insulin and insulin-like growth factor-I receptor precursors as well as a variety of viral envelope glycoprotein precursors.

Amino Acid Sequence↗

PubMed1990

Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain.

In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).

Amino Acid Sequence↗

PubMed1986

Cell-type-specific fibronectin subunits generated by alternative splicing.

Multiple fibronectin mRNAs arise by alternative splicing of the primary transcript of a single gene. We describe analyses of the contribution of this alternative splicing to fibronectin subunit heterogeneity in three different cell types using antisera directed against specific segments of fibronectin. beta-galactosidase-fibronectin fusion proteins produced with the lambda gt11 bacterial expression vector were used as immunogens. One region of alternative splicing accounts for differences in subunit size, while a second contributes to differences between the fibronectins present in blood plasma and in fibroblastic cells. We also show, however, that these two regions of alternative splicing do not account for all detectable subunits. We have also used these segment-specific antisera to show that blood platelets contain a spectrum of fibronectin subunits distinct from that found in blood plasma.

Animals↗

PubMed1985

On the origin of species of fibronectin.

Multiple different subunits of fibronectin are known to occur and their origin has been unclear. Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing suggested an explanation for some of this diversity of fibronectin subunits. Because the alternative splicing events occur within the coding region, the mRNAs differ in coding potential. We have prepared recombinant phage containing a rat fibronectin cDNA segment that is present in some fibronectin mRNAs and not in others. This segment was inserted in the beta-galatosidase gene of lambda gt11, and fusion protein produced by lysogens of the recombinant phage was purified and used as immunogen. The resulting antisera recognized some subunits of rat and hamster fibronectins but not others, indicating that inclusion or removal of this segment gives rise to mRNAs that encode different fibronectin subunits. In particular, presence or absence of a 95 amino acid segment appears to account for differences in size among the subunits of plasma fibronectin, whose origin is therefore explained by alternative patterns of RNA splicing.

Animals↗

PubMed1984

Multiple fibronectin subunits and their post-translational modifications.

We report analyses of fibronectin subunit diversity by high resolution one- and two-dimensional gel electrophoresis. We have studied plasma and cellular fibronectins of rats and hamsters. Each form of fibronectin comprises multiple distinguishable subunits and, within each rodent species, all subunits of plasma fibronectin are resolvable from those of cellular fibronectin. Some, but not all, of this heterogeneity is caused by differential glycosylation. Thus, while glycosylated plasma and cellular fibronectins share no common subunits, nonglycosylated forms of these proteins appear to share 2-3 subunits. In addition, there are subunits unique to plasma and to cellular fibronectins in both rats and hamsters, although the pattern of diversity differs slightly between species. All size variants of fibronectin are phosphorylated to varying degrees. However, only some subunits are sulfated, apparently on tyrosine residues in the C-terminal third of the molecule. Comparison of the distribution of sulfate on the various fibronectin subunits with recent results on generation of multiple mRNAs by alternative splicing suggests that tyrosine sulfate is located in a polypeptide segment present in only certain fibronectin subunits. The results reported here provide information on the likely contributions of primary sequence differences and post-translational modifications to the heterogeneity of fibronectin subunits.

Animals↗