Characterization and solubilization of the FMRFamide receptor of squid.
The optic lobe of squid (Loligo pealei) contains FMRFamide receptors that can bind an iodinated FMRFamide analog: [125I]-desaminoTyr-Phe- norLeu-Arg-Phe-amide ([125I]-daYFnLRFa). Radioligand binding assays revealed that squid FMRFamide receptors are specific, saturable, high affinity sites (Kd = 0.15 nM) densely concentrated in optic lobe membranes (Bmax = 237 fmole/mg protein). The receptors appeared to be coupled to Gs because guanine nucleotides inhibit receptor binding and the stimulation of adenylate cyclase by FMRFamide is GTP-dependent. Both the binding and cyclase data showed that FMRFamide, but not FMRF-OH, interacts at FMRFamide receptors; thus the C-terminal Arg-Phe-amide is critical for binding. The high binding affinity of FMRFamide (0.4 nM IC50) was specific for FMRFamide-like peptides. The structure-activity relations of many FMRFamide analogs were defined in detail and were nearly identical for both the membrane-bound and detergent-solubilized receptors. We also found that squid optic lobe contains FMRFamide-like reactivity as measured with both a radioimmunoassay and a radioreceptor assay. Moreover, we have sequenced a fragment of genomic DNA that encodes a FMRFamide precursor. Our findings in sum suggest that FMRFamide is a neurotransmitter in squid optic lobe, and that this tissue is a good source from which to purify FMRFamide receptors.
Chin
G J
GJ
Laboratory of Cellular and Molecular Neurophysiology, NICHD, Bethesda, Maryland 20892.
Payza
K
K
Price
D A
DA
Greenberg
M J
MJ
Doble
K E
KE
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000032:analysis; D049832:Decapodiformes; D019835:FMRFamide; D019204:GTP-Binding Proteins / Q000502:physiology; D006150:Guanine Nucleotides / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D009899:Optic Lobe, Nonmammalian / Q000737:chemistry / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics; D018010:Receptors, Invertebrate Peptide / Q000378:metabolism*; D012995:Solubility
C055209:FMRFamide receptor; D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D009479:Neuropeptides; D009842:Oligopeptides; D011498:Protein Precursors; D018010:Receptors, Invertebrate Peptide; C091318:desamino-tyrosyl-phenylalanyl-norleucyl-arginyl-phenylalaninamide; D019835:FMRFamide; D004247:DNA; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases