Epitope-tagged human GPCR and RAMP DNA constructs were encoded in a pcDNA3.1(+) mammalian expression vector. The human RAMP1, RAMP2, and RAMP3 complementary DNAs (cDNAs) encoded an N-terminal FLAG tag (DYKDDDDK) following the signal sequence (amino acids 1 to 26, 1 to 42, and 1 to 27 for RAMP1, RAMP2, and RAMP3 respectively) and a C-terminal OLLAS tag (SGFANELGPRLMGK). The human secretin-like GPCR and ADGRF5 cDNAs encoded the 23–amino acid residue 5-hydroxytryptamine receptor 3a receptor (5-HT3a) signal sequence (MRLCIPQVLLALFLSMLTGPGEG) in place of the native signal sequence, which was determined using SignalP 4.1 (Table 2) (40). In addition, an N-terminal HA tag (YPYDVPDYA) followed the 5HT3a signal sequence, and the C terminus encoded a 1D4 tag (TETSQVAPA). The RAMP, secretin-like GPCR, and ADGRF5 DNA constructs were codon optimized for expression in human cell lines. Expression constructs of human GPR182 and GPR4 cDNAs encoded an N-terminal HA tag (cDNA.org). The human CCR5, CCR7, and CXCR4 cDNAs encoded a C-terminal 1D4 epitope tag.

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