G protein coupled receptor transcripts in human immune cells and platelets

Abstract

G-protein coupled receptors (GPCRs) are encoded by nonabundant mRNAs, and it is difficult to detect them reliably with the highly parallel methods that are in general use. Because of this, we developed and validated a sensitive, specific, semi-quantitative method for detecting these transcripts. We have used the method to profile GPCR transcripts in white blood cells (WBCs)-B, CD4, CD8, NK, and dendritic cells; monocytes, and macrophage-like monocytes treated with granulocyte-macrophage colony-stimulating factor-as well as platelets. On average, the white cells studied expressed 160 receptor mRNAs (range, 123-206). Platelets made 69. Some, but far from all, of the receptors we found have been detected earlier. We believe our data should stimulate studies of receptor function and contribute to drug development.

Fig. 1

Experimental workflow. PBMCs were collected from healthy donors and sorted into different cell populations. RNA was prepared and its quality and concentration assayed according to standardized techniques. A total of 100 ng per reaction were used as input material to amplify GPCR transcripts in eight separate reactions each containing 50 GPCR specific primer pairs and aminoallyl-dUTP. Separate reactions with 10 ng of input material were used to determine those receptors expressed at higher levels. PCR reactions were pooled and Cy5 labelled on the amino-modified nucleotides. Labelled PCR products were hybridized to custom GPCR arrays that contained plus and minus sense oligonucleotides. Arrays were processed and scanned, and their signal extracted analyzed.

Fig. 2

Analysis of individual GPCR expression overlaps. Bars indicate the number of GPCR shared by the cell types indicated by connected black dots in the cell matrix. The total amount of GPCRs expressed by each cell type is indicated by the horizontal bars in the lower left quadrant.

Fig. 3

GPCR expression detection method comparison. (A) Venn analysis was used to compare the sensitivity of our method compared to the Groot et al. study at a Ct threshold of 35 cycles. Us-assayed means the GPCRs assayed in our study. Us-detected, the GPCRs detected in our 100 ng input assay. Groot-assayed, the total amount of GPCRs assayed by Groot-Kormelink et al. Groot-35 the GPCRs detected by TaqMan QPCR assays at a 35-cycle threshold in that study. (B) Venn analysis was used to compare the sensitivity of our methods compared to the Groot et al. study at a Ct threshold of 32 cycles. Us-assayed, the GPCRs assayed in our study. Us-detected, the GPCRs detected in our 100 ng input assay. Groot-assayed, the total amount of GPCRs assayed by Groot-Kormelink et al. Groot-32 the GPCRs detected by TaqMan QPCR assays at a 32-cycle threshold in that study.