Functional coupling with Galpha(q) and Galpha(i1) protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli.
To analyze the coupling of Galpha subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Galpha subunits covalently linked to the receptor C-terminus. The presence of Galpha(q) or Galpha(i/q), in which the six C-terminal residues of Galpha(i1) were replaced with those from Galpha(q), increased the percentage of receptors in the agonist high-affinity state. This effect was less pronounced for wild-type Galpha(i1) and not observed for Galpha(i/s). Functional coupling of neurotensin receptor to Galpha was demonstrated by neurotensin-induced [(35)S]GTPgammaS binding for the Galpha(q), Galpha(i/q) and Galpha(i1) subunits, but not for Galpha(i/s). Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.
Grisshammer
R
R
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. rkg@mrc-lmb.cam.ac.uk
Hermans
E
E
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits; D051381:Rats; D018028:Receptors, Neurotensin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins