title: Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I.
abstract: The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr(20) → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K(d) = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr(253) → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide ((505)QEAKL(509)) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr(253) → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.
authors: Department of Pathology, Harvard Medical School, Boston, Massachusetts 02215, USA. okocher@bidmc.harvard.edu
authors: Olivier
authors: O
authors: Kocher
authors: Gabriel
authors: G
authors: Birrane
authors: Ayce
authors: A
authors: Yesilaltay
authors: Sharon
authors: S
authors: Shechter
authors: Rinku
authors: R
authors: Pal
authors: Kathleen
authors: K
authors: Daniels
authors: Monty
authors: M
authors: Krieger
references: Curr Opin Lipidol. 2009 Jun;20(3):236-41
references: 19421056
references: Biochemistry. 2009 Mar 17;48(10):2261-71
references: 19173579
references: J Biol Chem. 2008 Aug 8;283(32):22097-104
references: 18544532
references: Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12610-5
references: 9356497
references: Methods Enzymol. 1997;276:307-26
references: 27754618
references: Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9322-7
references: 10430941
references: J Biol Chem. 2003 Dec 26;278(52):52820-5
references: 14551195
references: Protein Eng. 1995 Feb;8(2):127-34
references: 7630882
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6538-43
references: 10829064
references: Science. 1992 Oct 16;258(5081):468-71
references: 1411543
references: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references: 19461840
references: J Biol Chem. 2001 Jun 8;276(23):19683-6
references: 11304524
references: Cell. 1992 Oct 16;71(2):343-53
references: 1423598
references: Science. 2003 Apr 18;300(5618):445-52
references: 12702867
references: Biochim Biophys Acta. 2008 May;1782(5):310-6
references: 18342019
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2184-95
references: 15572771
references: J Biol Chem. 2002 Sep 13;277(37):34042-7
references: 12119305
references: J Biol Chem. 2010 Nov 5;285(45):34999-5010
references: 20739281
references: Endocr Rev. 2003 Jun;24(3):357-87
references: 12788804
references: Circ Res. 2002 Feb 22;90(3):270-6
references: 11861414
references: Annu Rev Genet. 1986;20:465-99
references: 3545063
references: Cell. 1996 Jun 28;85(7):1067-76
references: 8674113
references: FEBS J. 2006 Nov;273(22):5219-29
references: 17069616
references: Mol Biol Rep. 2003 Jun;30(2):69-82
references: 12841577
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references: 15572765
references: Curr Opin Lipidol. 2005 Apr;16(2):147-52
references: 15767854
references: J Biol Chem. 1996 May 10;271(19):11191-6
references: 8626666
references: J Biol Chem. 2009 Feb 27;284(9):5797-806
references: 19116202
references: PLoS One. 2009 Dec 01;4(12):e8103
references: 19956623
references: Mol Cell Biol. 2003 Feb;23(4):1175-80
references: 12556478
references: Acta Crystallogr A. 2008 Jan;64(Pt 1):112-22
references: 18156677
references: J Biol Chem. 2006 Sep 29;281(39):28975-80
references: 16867981
references: J Biol Chem. 1993 Apr 15;268(11):8221-9
references: 7681840
references: J Biol Chem. 2002 May 24;277(21):18973-8
references: 11882663