Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3.
The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.
Maile
Laura A
LA
Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 27599-7170, USA. laura_maile@med.unc.edu
Busby
Walker H
WH
Sitko
Kevin
K
Capps
Byron E
BE
Sergent
Tiffany
T
Badley-Clarke
Jane
J
Clemmons
David R
DR
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000906:Antibodies / Q000494:pharmacology; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D039661:Integrin beta3 / Q000378:metabolism*; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019096:Vitronectin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; C061248:ITGB3 protein, human; D039302:Integrin alphaVbeta3; D039661:Integrin beta3; D008024:Ligands; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019096:Vitronectin; D007334:Insulin-Like Growth Factor I; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3