Lysophosphatidylcholines activate G2A inducing G(αi)₋₁-/G(αq/)₁₁- Ca²(+) flux, G(βγ)-Hck activation and clathrin/β-arrestin-1/GRK6 recruitment in PMNs.
Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca²(+) via release of G(α) and G(βγ) subunits, kinase activation, and the recruitment of clathrin, β-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5-180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in G(αi)₋₁ and G(αq/)₁₁ release leading to increases in cytosolic Ca²(+), which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the G(βγ) subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr⁴¹¹). Moreover, G2A recruited clathrin, β-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and β-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of G(αi)₋₁, G(αq/)₁₁ and G(βγ), resulting in cytosolic Ca²(+) flux, Hck activation, and recruitment of clathrin, β-arrestin-1 and GRK6.
Khan
Samina Y
SY
Bonfils Blood Center, Denver, CO 80230, USA.
McLaughlin
Nathan J D
NJ
Kelher
Marguerite R
MR
Eckels
Phillip
P
Gamboni-Robertson
Fabia
F
Banerjee
Anirban
A
Silliman
Christopher C
CC
D019390:Arrestins / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D008856:Microscopy, Fluorescence / Q000379:methods; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; D019390:Arrestins; D018797:Cell Cycle Proteins; D002966:Clathrin; C115270:G2A receptor; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008244:Lysophosphatidylcholines; D043562:Receptors, G-Protein-Coupled; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D011494:Protein Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium
J Leukoc Biol. 2003 Apr;73(4):511-24
12660226
J Immunol. 2005 Mar 1;174(5):2981-9
15728511
Science. 1995 Jul 14;269(5221):221-3
7618083
Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):245-50
14681556
Biochem J. 1992 Dec 1;288 ( Pt 2):357-60
1334405
Nat Immunol. 2000 Sep;1(3):227-33
10973280
J Biol Chem. 1999 Jan 22;274(4):1957-65
9890951
Curr Opin Cell Biol. 2004 Apr;16(2):162-8
15196559
J Immunol. 2007 May 15;178(10):6540-8
17475884
J Biol Chem. 2006 Oct 20;281(42):31940-9
16920721
Blood. 1995 Sep 1;86(5):1649-60
7654998
J Leukoc Biol. 2006 Dec;80(6):1214-21
16943386
Proc Natl Acad Sci U S A. 1988 Apr;85(8):2805-9
3357891
J Clin Invest. 1998 Apr 1;101(7):1458-67
9525989
Clin Chem. 2006 Jan;52(1):19-29
16269514
Science. 2005 Apr 22;308(5721):512-7
15845844
J Lipid Res. 2005 Apr;46(4):623-7
15687351
Science. 2007 Feb 2;315(5812):605-6
17272707
J Biol Chem. 1994 Jul 29;269(30):19313-20
8034695
J Biol Chem. 2006 Jul 21;281(29):20577-88
16687412
J Lipid Res. 2001 Sep;42(9):1430-7
11518762
J Clin Invest. 1986 Jul;78(1):271-80
3013938
J Immunol. 2008 Jun 15;180(12):8192-203
18523285
FASEB J. 1994 May;8(8):504-12
8181668
Blood. 2003 Jan 15;101(2):454-62
12393667
Transfusion. 2003 May;43(5):633-40
12702186
J Cell Sci. 2002 Feb 1;115(Pt 3):455-65
11861753
J Biol Chem. 1995 Nov 24;270(47):28010-3
7499283
J Immunol. 2006 Jul 1;177(1):604-11
16785558
J Immunol. 2003 Dec 1;171(11):6128-34
14634128
J Lab Clin Med. 1994 Nov;124(5):684-94
7964126
J Cardiovasc Pharmacol. 1991;17 Suppl 7:S146-9
1725313
Sci STKE. 2004 Jan 27;2004(218):re3
14762218
Nat Med. 2004 Feb;10(2):161-7
14716308
J Immunol. 2006 Jun 1;176(11):7039-50
16709866
Nature. 1992 Dec 17;360(6405):686-9
1465134
Blood. 1994 Oct 1;84(7):2068-101
7522621
Am J Physiol Cell Physiol. 2002 Dec;283(6):C1592-603
12388071
Blood. 2006 Nov 1;108(9):2906-13
16849637
J Leukoc Biol. 2004 Apr;75(4):698-704
14704365
Curr Opin Cell Biol. 1999 Apr;11(2):177-83
10209148
Blood. 1992 Aug 15;80(4):1052-7
1323344
J Immunol. 2007 Mar 15;178(6):3874-85
17339487
Physiol Rev. 1998 Jan;78(1):189-225
9457173
J Biol Chem. 2006 Nov 24;281(47):36411-9
17008309
Nature. 2003 Mar 6;422(6927):37-44
12621426
J Clin Invest. 1994 Aug;94(2):815-23
8040337
J Biol Chem. 1987 Jan 15;262(2):589-94
3100516
Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2964-8
8610151
Am J Physiol Cell Physiol. 2003 Mar;284(3):C640-8
12431910