Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.
A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by alpha-subunits of the Gq family and by G-protein beta gamma-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-beta family, with the highest identity (71%) shared with PLC-beta 2 and lesser identities observed with PLC-beta 1 (49%), PLC-beta 3 (46%) and PLC-beta 4 (37%). The largest differences in sequence between the turkey PLC-beta and other PLC-beta isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-beta 2, which differ in their regulation by G-protein alpha-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in alpha q activation of these proteins. Recombinant turkey PLC-beta was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-beta. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-beta in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with G alpha 11 or G-protein beta gamma-subunits. The turkey PLC-beta was more sensitive to activation by G alpha 11 than was PLC-beta 2, and was more sensitive to activation by beta gamma-subunits than either PLC-beta 2 or PLC-beta 1.
Waldo
G L
GL
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7365, USA.
Paterson
A
A
Boyer
J L
JL
Nicholas
R A
RA
Harden
T K
TK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D054799:Phospholipase C beta; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism; D014422:Turkeys / Q000097:blood*; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D014357:Trypsin; D019204:GTP-Binding Proteins; D002118:Calcium