Critical functional requirement for the guanidinium group of the arginine 41 side chain of human epidermal growth factor as revealed by mutagenic inactivation and chemical reactivation.

In a preliminary study we demonstrated that the formation of the epidermal growth factor (EGF) receptor-ligand complex requires the participation of the highly conserved arginine 41 side chain of the growth factor peptide (Engler, D.A., Montelione, G.T., and Niyogi, S.K. (1990) FEBS Lett. 271, 47-50). In an attempt to gain further insight into the nature of this interaction(s), we used both site-directed mutagenesis and chemical modification reagents to produce human EGF (hEGF) analogues with altered chemical properties of the residue 41 side chain. Eight mutant analogues of hEGF were generated, substituting arginine 41 with lysine, glutamine, isoleucine, tyrosine, glycine, alanine, aspartate, or glutamate. Although each of the mutant analogues was able to displace wild-type hEGF fully in receptor competition binding assays, affinity of the receptor for the mutants was substantially reduced, varying from 0.4 to less than 0.01% of that observed for wild-type growth factor. At sufficiently high concentrations these mutants were able to stimulate DNA synthesis in mouse keratinocytes. Substitution of lysine for arginine 41 reduced the receptor affinity 250-fold from that observed for wild type, despite retention of the positive electrostatic charge. The lysine substitution leaves a reactive amine at position 41 and made it possible, using amine-specific chemical modification reagents, to produce selected arginine homologues that were tested for their effects on receptor binding, receptor tyrosine kinase activation, and stimulation of DNA synthesis in mouse keratinocytes. The reaction of lysine 41 with methyl acetimidate resulted in a lysineacetamidine product which only partially restored activity of the lysine hEGF mutant. However, reaction with O-methylisourea resulted in generation of an arginine 41 homologue (homoarginine) which restored full activity. The results indicate that the chemical properties inherent in the guanidinium group of the arginine 41 side chain of hEGF are responsible for optimal receptor-ligand association.

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native [125I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe greater than His greater than Ser greater than Ala greater than Asp greater than Arg greater than Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37----Ala, Tyr-37----Arg and Tyr-37----Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.

Uptake and metabolism of putrescine in confluent LLC-PK1 cells.

Putrescine is taken up by confluent pig kidney (LLC-PK1) cells at roughly equal rates over both Na(+)-dependent and Na(+)-independent pathways. The former is sensitive to 1 mM amiloride, but the latter is not. Uptake rates are similar at both the apical and basolateral surfaces. The principal fate of the putrescine is oxidative deamination, yielding a product that appears to be either gamma-aminobutyraldehyde or delta 1-pyrroline. Most of the remainder is converted to products tentatively identified as spermidine, spermine, or another unidentified product; these products as well as putrescine itself are lost from the cell at either surface. Changing the extracellular pH in the range of 6.8-8.0 has no affect on putrescine uptake. Cells acidified to intracellular pH 6.8 show a reduced capacity to incorporate radioactivity, an effect that may be due to inhibition of diamine oxidase. Depletion of ATP stores by treating cells with 2-deoxy-D-glucose and NaN3 does not reduce putrescine uptake, suggesting that the mechanism is not a primary active transporter. The Na(+)-dependent component of uptake is inhibited by 5-50 microM Hg2+ in a dose-dependent manner. p-Chloromercuribenzene sulfonic acid (p-CMBS) at high concentrations (500-1,000 microM) does not affect Na(+)-independent uptake but in the presence of Na+ depresses total uptake more than Na+ depletion alone, suggesting that Na+ enhances the binding of p-CMBS to both transporters. Spermidine and spermine compete with putrescine for uptake, but a variety of other organic bases and amino acids do not, indicating that polyamines are transported by mechanisms distinct from the transporters for those other compounds.