Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.
G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.
Shi
Mei
M
Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Bennett
Teresa A
TA
Cimino
Daniel F
DF
Maestas
Diane C
DC
Foutz
Terry D
TD
Gurevich
Vsevolod V
VV
Sklar
Larry A
LA
Prossnitz
Eric R
ER
D000818:Animals; D019393:Arrestin / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020298:U937 Cells; D014461:Ultracentrifugation / Q000379:methods
D019393:Arrestin; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498016:Gnai2 protein, mouse; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium