Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor.

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.

Calorimetry of cholinesterase activity in serum.

Cholinesterase activity in human serum was measured calorimetrically and compared to values obtained with the spectrophotometric method of Rappaport et al. [Clin. Chim. Acta 4, 227 (1959)]. The response of the calorimeter upon mixing serum with acetylcholine corresponded to the cholinesterase activity of the serum, the thermogram peak height being linearly related to enzyme activity at serum dilution volumes at which interference heat was negligible. The interference heat contributed by the dilution mixing characteristics of serum, studied separately, was found to be similar for heat-denatured, aged, or lyophilized samples. In all cases, heat of mixing was negligible when 1 ml of serum in buffer (2/3 by vol) was mixed with 1 ml of buffer. When this amount of serum was assayed with acetylcholine, cholinesterase activities of 2 and 3 kU/liter were recorded as thermograms with peak heights of 2.3 and 3.5 cm, respectively.