Cell adhesion receptor GPR133 couples to Gs protein

Abstract

Adhesion G protein-coupled receptors (GPCR), with their very large and complex N termini, are thought to participate in cell-cell and cell-matrix interactions and appear to be highly relevant in several developmental processes. Their intracellular signaling is still poorly understood. Here we demonstrate that GPR133, a member of the adhesion GPCR subfamily, activates the G(s) protein/adenylyl cyclase pathway. The presence of the N terminus and the cleavage at the GPCR proteolysis site are not required for G protein signaling. G(s) protein coupling was verified by Gα(s) knockdown with siRNA, overexpression of Gα(s), co-expression of the chimeric Gq(s4) protein that routes GPR133 activity to the phospholipase C/inositol phosphate pathway, and missense mutation within the transmembrane domain that abolished receptor activity without changing cell surface expression. It is likely that not only GPR133 but also other adhesion GPCR signal via classical receptor/G protein-interaction.

FIGURE 1.

GPR133 increases cAMP accumulation in HEK293 and COS-7 cells. A, the domain architecture of the human GPR133 is shown. To monitor GPR133 expression, the receptor was N-terminally epitope-tagged downstream of the signal peptide (SP) with an HA epitope followed by a sequence encoding the N-terminal 20 amino acids of bovine rhodopsin N terminus (RHO) and C-terminally with a FLAG epitope (FLAG). B, HEK293 cells were transfected with the human and mouse GPR133 and a construct where the N terminus of the human GPR133 was replaced by the bovine rhodopsin N terminus (human RhoGPR133), and CRE-SEAP assays were performed as described under “Experimental Procedures.” Basal (transfected pcDps vector) SEAP activity was 155,634 ± 8,949 arbitrary units/well, and stimulation of cells with 5 μm forskolin resulted in a 4.89 ± 2.84-fold increase in SEAP activity. Data are given as means ± S.D. of two independent experiments performed in quadruplicates. C, COS-7 cells were transfected with increasing amounts (0, 200, 300, 400 ng/well) of plasmid encoding the human GPR133. After 2 days, intracellular cAMP levels and cell surface expression levels were determined as described under “Experimental Procedures.” The pcDps vector served as negative control showing a cAMP level of 6.3 ± 1.5 nm/well and an optical density (OD) value of 0.012 ± 0.001 OD_{492–620 nm}. Data of a representative assay are given as means ± S.D. (-fold over negative control) performed in triplicate. D, COS-7 cells were transfected with the wild-type (wt) and human GPR133 mutants. Two days after transfection, intracellular cAMP levels (D) and cellular expression levels (E) were determined as described under “Experimental Procedures.” Cyclic AMP levels were referred to the negative control (pcDps; cAMP level: 21.9 ± 3.2 nm/well). Data are given as means ± S.E. (-fold over negative control) of three independent experiments each performed in triplicate. For expression studies, cell surface and sandwich ELISA were used to measure cell surface and total cellular expression levels, respectively. Specific OD readings (OD value of double HA/FLAG-tagged GPR133 constructs minus OD value of mock-transfected cells) are given as the percentage of double HA/FLAG-tagged WT GPR133. For the cell surface ELISA, the nonspecific OD value (pcDps) was 0.040 ± 0.003 (set as 0%), and the OD value of WT GPR133 was 1.091 ± 0.323 (set as 100%). OD readings of 0.022 ± 0.002 (set as 0%) and 1.149 ± 0.042 (set as 100%) were found in sandwich ELISA (total expression) for the negative control vector (pcDps) and the WT GPR133, respectively. Data are given as means ± S.E. of three independent experiments each performed in triplicate.

FIGURE 2.

GPR133 is coupled to the G_s protein. A, to evaluate the signaling specificity of GPR133, COS-7 cells were co-transfected with the indicated receptor construct and the chimeric Gα_qs4 protein (see “Experimental Procedures”). The Gα_s-coupled MC4R served as positive control and was stimulated with 10 μm αMSH. IP assays were performed 48 h after transfection as described under “Experimental Procedures.” Basal IP formation is expressed as X-fold over basal levels of mock-transfected cells (204 ± 52 cpm/well). Data are presented as means ± S.D. of four (GPR133) and two (MC4R) independent experiments, each carried out in triplicate. ***, p < 0.001. B, COS-7 cells were co-transfected with 225 ng/well of plasmid encoding the human GPR133 or vector control (pcDps) and increasing amounts (0, 7.5, 15, 30, 45, 60, 75 ng/well) of plasmid encoding the human Gα_s. Data are given as means ± S.E. (-fold over negative control) of three independent experiments each performed in triplicate. C, in a second setup, COS-7 cells were co-transfected with 60 ng/well of plasmid encoding human Gα_s or vector control (pcDps) and increasing amounts (0, 25, 75, 125, 175, 225 ng/well) of plasmid encoding the human GPR133. After 2 days, intracellular cAMP levels were determined as described under “Experimental Procedures.” The empty pcDps vector served as negative control (cAMP level: 3.1 ± 0.9 nm/well). Data are given as means ± S.E. (-fold over negative control) of three independent experiments each performed in triplicate. D, HEK293 cells were co-transfected with 600 ng/well of plasmid encoding the human GPR133 in pcDNA3.1 or vector control (pcDNA3.1) and 2.5 pmol/well Gα_s siRNA or control siRNA. The human cell line HEK293 was used to meet the species specificity of the siRNA against the human Gα_s subunit. After 2 days, intracellular cAMP levels were determined as described under “Experimental Procedures.” The vector pcDNA3.1 served as negative control (cAMP level: 6.1 ± 0.6 nm/well). Data are given as means ± S.E. (-fold over negative control) of three independent experiments each performed in triplicate.