Incorporation of Galpha(z)-specific sequence at the carboxyl terminus increases the promiscuity of galpha(16) toward G(i)-coupled receptors.
Although the promiscuous nature of G(16) allows it to interact with numerous G protein-coupled receptors, several G(i)-linked receptors are incapable of activating phospholipase C via G(16). A series of chimeras between Galpha(16) and Galpha(z) were constructed and assayed for their ability to mediate receptor-induced stimulation of phospholipase C. Two Galpha(16/z) chimeras harboring 25 or 44 Galpha(z)-specific sequences at their C termini (named 16z25 and 16z44) were capable of responding to 14 different G(i)-coupled receptors tested, including those that were either unable to associate with Galpha(16) (melatonin Mel1c) or activate Galpha(16) weakly (micro-opioid and type 1 somatostatin). Agonist-induced stimulation of phospholipase C was more efficiently mediated (higher maximal and lower EC(50) value) by 16z44 than by Galpha(16). Both 16z25 and 16z44 were also coupled to G(s)- and G(q)-linked receptors. Incorporation of Galpha(z) sequence at the N terminus of Galpha(16) did not further enhance the ability of the chimeras to interact with G(i)-coupled receptors. Expression of the various chimeras was verified by immunodetection and functional analysis of their constitutively activated mutants. These results show that the incorporation of alpha4/beta6 and alpha5 regions of Galpha(z) into a Galpha(16) backbone can improve the recognition of G(i)-coupled receptors. Galpha(16/z) chimeras with expanded capability to interact with G(i)-linked receptors may be used to link orphan receptors to the stimulation of phospholipase C.
Mody
S M
SM
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Ho
M K
MK
Joshi
S A
SA
Wong
Y H
YH
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D044385:GTP-Binding Protein alpha Subunits; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D044385:GTP-Binding Protein alpha Subunits; D011956:Receptors, Cell Surface; D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins; C112631:G protein alpha 16; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins