Characterization and Solubilization of the FMRFamide Receptor of Squid

• Published in: The Biological bulletin (Lancaster) Vol. 187; no. 2; pp. 185 - 199
• Main Authors: Chin, Gilbert J., Payza, Kemal, Price, David A., Greenberg, Michael J., Doble, Karen E.
• Format: Journal Article
• Language: English
• Published: United States Marine Biological Laboratory 01.10.1994; University of Chicago Press
• Subjects: Adenylyl Cyclases - metabolism; Amino Acid Sequence; Animals; Antiserum; Biochemistry; Biology; Cell Membrane - metabolism; Chemoreceptors; Decapodiformes; DNA - analysis; FMRFamide; Generally accepted auditing standards; GTP-Binding Proteins - physiology; Guanine Nucleotides - pharmacology; Inhibitory concentration 50; Iodine Radioisotopes; Ligands; Molecular Sequence Data; Mollusks; Neurobiology; Neuropeptides; Neuropeptides - analysis; Neuropeptides - genetics; Neuropeptides - metabolism; Nucleotides; Oligopeptides - metabolism; Optic lobe; Optic Lobe, Nonmammalian - chemistry; Optic Lobe, Nonmammalian - metabolism; Optic nerve; Physiological aspects; Protein Precursors - genetics; Receptors; Receptors, Invertebrate Peptide - metabolism; Solubility; Solubilization; Squid; Squids
• ISSN: 0006-3185; 1939-8697
• DOI: 10.2307/1542241

The optic lobe of squid (Loligo pealei) contains FMRFamide receptors that can bind an iodinated FMRFamide analog: [125 I]-desaminoTyr-Phe-norLeu-Arg-Phe-amide ([125 I]-daYFnLRFa). Radioligand binding assays revealed that squid FMRFamide receptors are specific, saturable, high affinity sites ( K d=0.15 nM) densely concentrated in optic lobe membranes ( B max=237 fmole/ mg protein). The receptors appeared to be coupled to G s 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.