We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N‐terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild‐type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor‐catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector‐activating interaction we measured the affinity of [W207F]T alpha GDP‐AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation‐dependent way to PDE gamma, but with a 100‐fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events.

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.