Reconstitution reveals additional roles for N- and C-terminal domains of g(alpha) in muscarinic receptor coupling.
The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.
Slessareva
Janna E
JE
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9223, USA.
Graber
Stephen G
SG
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D020662:Guanine Nucleotide Exchange Factors; D021122:Protein Subunits; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins