Palmitoylation but not the extreme amino-terminus of Gq alpha is required for coupling to the NK2 receptor.
Gq alpha and G11 alpha differ from other G protein alpha subunits in that they have unique, conserved 6 residue amino-terminal extensions. Wild-type and amino-terminal mutants of Gq alpha expressed in COS cells were analyzed for their ability to functionally couple with co-expressed neurokinin NK2 receptor. Wild-type, T2A and delta 2-7 Gq alpha were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino-terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with G beta gamma subunits and GTP gamma S-induced trypsin resistance are also similar to the wild-type alpha subunit. This demonstrates that the NK2 receptor is able to functionally interact with the alpha subunit of Gq and that the first seven amino-acids of Gq alpha are not required for any of the alpha subunit functions tested. In contrast to the T2A and delta 2-7 mutants, a C9,10A Gq alpha mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high-affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTP gamma S-induced trypsin-resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild-type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino-terminus of Gq alpha results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
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MD
Edgerton
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C
Chabert
A
A
Chollet
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S
Arkinstall
D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism; D003902:Detergents; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D016296:Mutagenesis; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010957:Plasmids; D018041:Receptors, Neurokinin-2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D017607:Aluminum Compounds; D003567:Cytidine Diphosphate Diglycerides; D003902:Detergents; D010169:Palmitic Acids; D018041:Receptors, Neurokinin-2; D011994:Recombinant Proteins; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride