RESUMO
EGF activates the ErbB1 receptor, but there appears only a limited correlation between its receptor binding affinity and mitogenic activity. This is indicated by our present observation that in cells with high ErbB1 expression, including SUM102 breast tumor cells, low affinity EGF/Notch chimeras have similarly high mitogenic activity as EGF, in spite of the fact that EGF is superior in inducing receptor tyrosine phosphorylation and p42/p44 MAP-kinase activity. However, as a result of receptor-mediated internalisation high-affinity ligands such as EGF are depleted much more rapidly from the extracellular medium than low-affinity EGF/Notch chimeras. As a consequence, the mitogenic activity of EGF on ErbB1 overexpressing cells is limited by substantial degradation of internalised ligand in the period before cells enter S-phase, a phenomenon that is not observed for low affinity mutant ligands. The mitogenic activity of EGF on ErbB1 overexpressing cells does therefore not only depend on the applied concentration but also on the total amount of ligand added, and is strongly underestimated when tested in a limited assay volume. No such dependence on the incubation volume was observed for EGF activity on cells with low ErbB1 expression levels and on cells for which EGF is growth inhibitory.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Retroalimentação Fisiológica/fisiologia , Substâncias de Crescimento/metabolismo , Mitose/fisiologia , Neoplasias/metabolismo , Agregação de Receptores/genética , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Feminino , Substâncias de Crescimento/genética , Humanos , Ligantes , Camundongos , Mitose/efeitos dos fármacos , Mutação/genética , Células NIH 3T3 , Neoplasias/genética , Ligação Proteica/fisiologia , Agregação de Receptores/efeitos dos fármacos , Receptor Notch1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fase S/efeitos dos fármacos , Fase S/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologiaRESUMO
Epidermal growth factor (EGF) binds with high affinity to the EGF receptor, also known as ErbB-1, but upon replacement of the N-terminal linear region by neuregulin (NRG) 1 or transforming growth factor (TGF) alpha sequences it gains in addition high affinity for ErbB-2/ErbB-3 heterodimers. However, these chimeras weakly bind to ErbB-3 alone. To further dissect the ligand binding selectivity of the ErbB network, we have applied the phage display technique to examine the role of the linear N-terminal region in EGF for interaction with ErbB-2/ErbB-3 heterodimers. A library of EGF variants was constructed in which residues 2, 3, and 4 were randomly mutated, followed by selection for binding to intact MDA-MB-453 cells that overexpress ErbB-2 and ErbB-3 but lack ErbB-1. Analysis of the selected phage EGF variants revealed clones with high binding affinity to ErbB-2/ErbB-3 while maintaining high affinity to ErbB-1. In these variants, Trp (or alternatively His) was almost exclusively present at position 2, while specific combinations of hydrophobic, basic, and small residues were found at positions 3 and 4. The mitogenic activity of the phage EGF variants corresponded with their relative binding affinity. Two of the selected EGF variants, EGF/WVS and EGF/WRS, were further characterized as recombinant proteins. In contrast to previously characterized chimeras of EGF with NRG-1 or TGF-alpha, these variants did not only show high binding affinity for ErbB-2/ErbB-3 heterodimers but also for ErbB-3 alone. These data show that the linear N-terminal region of EGF-like growth factors is directly involved in binding to ErbB-3.
Assuntos
Fator de Crescimento Epidérmico/química , Receptor ErbB-2/química , Receptor ErbB-3/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Neoplasias da Mama , Primers do DNA , Dimerização , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Feminino , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas Recombinantes/química , Células Tumorais CultivadasRESUMO
Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.