The beta6/alpha5 regions of Galphai2 and GalphaoA increase the promiscuity of Galpha16 but are insufficient for pertussis toxin-catalyzed ADP-ribosylation.
Replacement of beta6/alpha5 region at the C-terminus on Galpha16 with Galphaz-specific residues has been shown to broaden the promiscuity of Galpha16. Here, we substituted the last 44 residues of Galpha16 with the corresponding region from either Galphai2 or GalphaoA (16i44 and 16o44). 16i44 and 16o44 chimeras were more effective than Galpha16 at coupling to Gi-linked delta-opioid, mu-opioid, and Xenopus melatonin MT1c receptors when coexpressed in green monkey fibroblast (COS-7) cells. 16i44, but not 16o44, also enhanced the formyl peptide-induced stimulation of phospholipase C activity. Both chimeras were resistant to pertussis toxin-catalyzed [32P]ADP-ribosylation, despite the fact that pertussis toxin partially inhibited the chimera-mediated stimulation of phospholipase Cbeta. The use of Galphat1 as a Gbetagamma scavenger revealed that the pertussis toxin-sensitivity can be attributed to endogenous Gbetagamma subunits released from G(i/o). Although incorporation of a Galphai-like beta6/alpha5 region into the C-terminus of Galpha16 increases its promiscuity, this region is not sufficient to support recognition by pertussis toxin.
Wong
Cecilia S S
CS
Department of Biochemistry, Molecular Neuroscience Center, and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Ho
Maurice K C
MK
Wong
Yung H
YH
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D037342:Pertussis Toxin / Q000494:pharmacology*; D054799:Phospholipase C beta; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D044094:Receptors, Melatonin / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014981:Xenopus
D007295:Inositol Phosphates; D007527:Isoenzymes; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D044094:Receptors, Melatonin; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C112631:G protein alpha 16; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C498016:Gnai2 protein, mouse; C498017:Gnai2 protein, rat; D020962:Heterotrimeric GTP-Binding Proteins