Comparative Assessment of the Ligand and Metal Ion Binding Properties of Integrins 9
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- 作者：R. Blake Pepinsky ; Richard A. Mumford ; Ling Ling Chen ; Diane Leone ; Suzanne E. Amo ; Gail Van Riper ; Adrian Whitty ; Brian Dolinski ; Roy R. Lobb ; Dennis C. Dean ; Linda L. Chang ; Conrad E. Raab ; Qian Si
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：June 4, 2002
- 年：2002
- 卷：41
- 期：22
- 页码：7125 - 7141
- 全文大小：245K
- 年卷期：v.41,no.22(June 4, 2002)
- ISSN：1520-4995
文摘
Integrins 
9
1 and 41 form a distinct structural class, but while 41 has been subjectedto extensive study, 91 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical propertiesof 91 and directly compare these properties with those of 41. Compound 3 has a high affinity forboth integrins with KD values of 
3 and 180 pM for 91 in 1 mM Mn2+(activating) and 1 mM Ca2+and 1 mM Mg2+ (nonactivating) conditions and 5 and 730 pM for 41 under the correspondingconditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 
M Ca2+ in bothcases). Compound 3 binding to both integrins was also stimulated by the addition of the activatingmonoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions andTS2/16 regulate ligand binding to 91 and 41 are similar. The binding of 3 to both integrins inducedthe mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding siteby 3. But whereas EGTA treatment inhibited the binding of 9EG7 to 41, it stimulated the binding of9EG7 to 91. The 9EG7 and TS2/16 effects point to contributions of the 1-chains on binding. Cross-linking data revealed that the integrin -chains are also involved in binding the small molecule, as stablelinkages were observed on both the 9 chain of 91 and the 4 chain of 41. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets.Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, whichbinds 41with an apparent KD of 10 nM and 91 with an apparent KD of >10 M. Differences werealso seen in the binding of 91 and 41 to osteopontin. Compound 3 competed effectively for thebinding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in thebiochemical properties of 91 and 41, they identify important differences in their structure and functionthat can be exploited in the design of selective 91 and 41 inhibitors.
91 and 41 form a distinct structural class, but while 41 has been subjectedto extensive study, 91 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical propertiesof 91 and directly compare these properties with those of 41. Compound 3 has a high affinity forboth integrins with KD values of 3 and 180 pM for 91 in 1 mM Mn2+(activating) and 1 mM Ca2+and 1 mM Mg2+ (nonactivating) conditions and 5 and 730 pM for 41 under the correspondingconditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 M Ca2+ in bothcases). Compound 3 binding to both integrins was also stimulated by the addition of the activatingmonoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions andTS2/16 regulate ligand binding to 91 and 41 are similar. The binding of 3 to both integrins inducedthe mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding siteby 3. But whereas EGTA treatment inhibited the binding of 9EG7 to 41, it stimulated the binding of9EG7 to 91. The 9EG7 and TS2/16 effects point to contributions of the 1-chains on binding. Cross-linking data revealed that the integrin -chains are also involved in binding the small molecule, as stablelinkages were observed on both the 9 chain of 91 and the 4 chain of 41. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets.Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, whichbinds 41with an apparent KD of 10 nM and 91 with an apparent KD of >10 M. Differences werealso seen in the binding of 91 and 41 to osteopontin. Compound 3 competed effectively for thebinding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in thebiochemical properties of 91 and 41, they identify important differences in their structure and functionthat can be exploited in the design of selective 91 and 41 inhibitors.