Ectodomain shedding, the proteolytic cleavage of an integral membrane protein to release the extracellular domain from the host cell, affects a variety of biologically important proteins including growth factor precursors, cytokine receptors, amyloid precursor proteins and cell adhesion receptors, and proteoglycans. Therefore malfunction of ectodomain shedding often leads to various diseases. The long- term objective of this project is to elucidate the molecular and structural basis for shedding regulation across the cell membrane. Intracellular proteins can regulate shedding by interacting directly with the cytoplasmic domain of the shedding protein substrate. The best characterized example is the calmodulin (CaM) association with L-selectin to inhibit shedding of L-selectin. One clue to the regulation mechanism is the well documented but unexplained observation that the distance between the membrane-proximal shedding cleavage site in L-selectin and the cell membrane, rather than the sequence at the cleavage site, is critical to shedding activity. Shortening the distance abolishes shedding. The membrane-proximal region of the L-selectin cytoplasmic domain interacts with CaM, but it is only 12 residues long, much shorter than a typical CaM-binding sequence. Our recent study on the interaction of CaM with L-selectin-derived peptides suggested that the CaM-binding region in L-selectin may also include a portion of the L-selectin transmembrane domain. We hypothesize that CaM interaction with L-selectin affects its transmembrane domain in the membrane, which in turn changes the conformation and/or accessibility of the shedding cleavage site on the other side of the membrane. A portion of the L-selectin transmembrane domain may, upon CaM association, partition into CaM, thereby moving the entire TM domain and shedding cleavage site toward the cytoplasm and shortening the distance between the shedding cleavage site and the membrane, and effectively inhibit shedding of L-selectin.
In Specific Aim 1, the energetic and structural basis for the interaction of CaM with water-soluble peptides derived from L-selectin will be further characterized.
In Specific Aim 2, the interaction of CaM with a L-selectin fragment in membrane-mimicking environments will be characterized with NMR and fluorescence spectroscopy. The fragment contains the shedding cleavage site, the transmembrane and cytoplasmic domains of L-selectin. The focus will be to detect any conformational and/or positional changes in the L-selectin transmembrane domain induced by CaM association.
In Specific Aim 3, changes at the shedding cleavage site in the L-selectin fragment induced by CaM association, including any changes in the distance between the shedding cleavage site and the membrane bilayer, will be characterized. A careful study of the complexes of CaM with the L- selectin peptides will help to elucidate the mechanism underlying CaM regulation of L-selectin shedding, and provide insights into shedding regulation mechanisms in general.

Public Health Relevance

Relevance to public health: Ectodomain shedding affects a variety of biologically important proteins. It malfunction often leads to diseases. Elucidating the mechanism underlying calmodulin regulation of L- selectin shedding will contribute to a better understanding of shedding regulation. Our findings may lead to novel therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084175-05
Application #
8207999
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2009-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
5
Fiscal Year
2012
Total Cost
$300,718
Indirect Cost
$104,698
Name
Emory University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Deng, Wei; Cho, Sungyun; Su, Pin-Chuan et al. (2014) Membrane-enabled dimerization of the intrinsically disordered cytoplasmic domain of ADAM10. Proc Natl Acad Sci U S A 111:15987-92
Deng, Wei; Putkey, John A; Li, Renhao (2013) Calmodulin adopts an extended conformation when interacting with L-selectin in membranes. PLoS One 8:e62861
Deng, Wei; Cho, Sungyun; Li, Renhao (2013) FERM domain of moesin desorbs the basic-rich cytoplasmic domain of l-selectin from the anionic membrane surface. J Mol Biol 425:3549-62
Srinivasan, Sankaranarayanan; Deng, Wei; Li, Renhao (2011) L-selectin transmembrane and cytoplasmic domains are monomeric in membranes. Biochim Biophys Acta 1808:1709-15
Deng, Wei; Srinivasan, Sankaranarayanan; Zheng, Xiaofeng et al. (2011) Interaction of calmodulin with L-selectin at the membrane interface: implication on the regulation of L-selectin shedding. J Mol Biol 411:220-33
Mo, Xi; Nguyen, Nam X; Mu, Fi-Tjen et al. (2010) Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha. J Biol Chem 285:32096-104