The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.
Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.
Kühn
B
B
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Gudermann
T
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D005822:Genetic Vectors / Q000378:metabolism; D006168:Guinea Pigs; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D011518:Proto-Oncogene Proteins; D011974:Receptors, LH; D011994:Recombinant Proteins; D000242:Cyclic AMP; D010738:Type C Phospholipases; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498016:Gnai2 protein, mouse; D020962:Heterotrimeric GTP-Binding Proteins