Enhancing integrin α1 inserted (I) domain affinity to ligand potentiates integrin α1β1-mediated down-regulation of collagen synthesis.
Integrin α1β1 binding to collagen IV, which is mediated by the α1-inserted (I) domain, down-regulates collagen synthesis. When unligated, a salt bridge between Arg(287) and Glu(317) is thought to keep this domain in a low affinity conformation. Ligand binding opens the salt bridge leading to a high-affinity conformation. How modulating integrin α1β1 affinity alters collagen homeostasis is unknown. To address this question, we utilized a thermolysin-derived product of the α1α2α1 network of collagen IV (α1α2α1(IV) truncated protomer) that selectively binds integrin α1β1. We show that an E317A substitution enhanced binding to the truncated protomer, consistent with a previous finding that this substitution eliminates the salt bridge. Surprisingly, we show that an R287A substitution did not alter binding, whereas R287E/E317R substitutions enhanced binding to the truncated protomer. NMR spectroscopy and molecular modeling suggested that eliminating the Glu(317) negative charge is sufficient to induce a conformational change toward the open state. Thus, the role played by Glu(317) is largely independent of the salt bridge. We further show that cells expressing E317A or R287E/E317R substitutions have enhanced down-regulation of collagen IV synthesis, which is mediated by the ERK/MAPK pathway. In conclusion, we have demonstrated that modulating the affinity of the extracellular α1 I domain to collagen IV enhances outside-in signaling by potentiating ERK activation and enhancing the down-regulation of collagen synthesis.
Department of Medicine (Nephrology), Vanderbilt University, Nashville, Tennessee 37232.
Mingjian
M
Shi
Department of Medicine (Nephrology), Vanderbilt University, Nashville, Tennessee 37232; Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232.
Vadim
V
Pedchenko
Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232.
Briana H
BH
Greer
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Wade D
WD
Van Horn
Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee 37232.
Samuel A
SA
Santoro
Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Charles R
CR
Sanders
Department of Medicine (Nephrology), Vanderbilt University, Nashville, Tennessee 37232; Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Billy G
BG
Hudson
Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Brandt F
BF
Eichman
Department of Medicine (Nephrology), Vanderbilt University, Nashville, Tennessee 37232; Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Medicine, Veterans Affairs Medical Center, Nashville, Tennessee 37212.
Roy
R
Zent
Department of Medicine (Nephrology), Vanderbilt University, Nashville, Tennessee 37232; Center for Matrix Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Medicine, Veterans Affairs Medical Center, Nashville, Tennessee 37212. Electronic address: ambra.pozzi@vanderbilt.edu.
Ambra
A
Pozzi
D019943:Amino Acid Substitution; D000818:Animals; D002461:Cell Line, Transformed; D024141:Collagen Type IV / Q000096:biosynthesis* / Q000235:genetics; D015536:Down-Regulation; D004789:Enzyme Activation / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D039222:Integrin alpha1beta1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007797:Laminin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D020935:MAP Kinase Signaling System; D051379:Mice; D008817:Mice, Mutant Strains; D008958:Models, Molecular; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary
D024141:Collagen Type IV; D039222:Integrin alpha1beta1; D007797:Laminin; D008024:Ligands; C084098:laminin A; D048049:Extracellular Signal-Regulated MAP Kinases