Mark W. Riemen

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.

Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.

Spontaneously Transformed NRK Cells Lose Their Mitogenic Response to Epidermal Growth Factor

To understand the relationship between growth factor-induced mitogenesis and spontaneous cell transformation, a clonal isolate of epidermal growth factor (EGF)-responsive NRK cells was passed in vitro until morphologically transformed variants arose. Subclones of EGF responsive (Cl-3) and EGF nonresponsive (Cl-10) NRK cells were isolated. Cl-3 cells grew as flat, contact-inhibited monolayers, while Cl-10 cells grew as rounded or spindle-shaped cells that formed dense foci. Cl-10 cells formed colonies in soft agar more efficiently (p less than 0.01) and formed larger tumors in nude mice (p less than 0.05) than Cl-3 cells. Cl-3 cells exhibited a sixfold increase in DNA synthesis in response to 1.0 nM EGF. Cl-10 cells did not increase DNA synthesis on exposure to 100 nM EGF. These different responses to EGF occurred despite similar numbers of receptors and similar receptor.binding affinities for EGF (Cl-3: 7000 receptors, Kd = 0.67 nM; Cl-10: 8000 receptors, Kd = 0.72 nM). No evidence of transforming growth factor-alpha was detected in either of these cell lines using Northern blots, Western blots, or biologic assays. We conclude that NRK cells which undergo spontaneous morphologic transformation and exhibit enhanced anchorage-independent growth lose their mitogenic response to EGF.

Minimal Ligand Analysis of Gastrin‐Releasing Peptide: Receptor Binding and Mitogenesis

Gastrin releasing peptide (GRP) is a peptide hormone containing 27 amino acids which is structurally analogous to the amphibian peptide bombesin. GRP serves a variety of physiological functions and has been implicated in the pathophysiology of small cell lung cancer. Previous work has demonstrated that the modified C terminus of GRP, N-acetyl-GRP-20-27, exerts full agonist activity in a variety of assay systems. However, no systematic comparison of binding of GRP fragments to its receptor and mitogenic potency has been reported. To investigate whether smaller GRP fragments could bind to the GRP receptor without stimulating mitogenesis, we performed binding inhibition and thymidine uptake assays with Swiss 3T3 fibroblasts. These studies were facilitated by the development of a novel tritiated GRP-based radioligand, [3H-Phe15] GRP-15-27, which exhibits enhanced chemical stability compared to iodinated GRP derivatives. We examined a series of C-terminal GRP fragments, from the pentapeptide to the octapeptide, with both N-acetyl and free amine moieties at the N terminus. N-Acetylated derivatives were more potent than their primary amine counterparts in both assays. Deletion of N-terminal residues from GRP-20-27 resulted in significant loss of potency in both assays: the EC50 values of N-acetyl-GRP-21-27 were 10(2)-fold higher than N-acetyl-GRP-20-27, those of N-acetyl-GRP-22-27 were 10(4)-fold higher, and N-acetyl-GRP-23-27 showed minimal activity at concentrations below 100 microM. These results suggest that 1) both His20 and Trp21 play an important role in binding of GRP to the receptor, and 2) for this series of N-terminal deletions, binding to the receptor and mitogenic activity are tightly coupled.