Octopamine- and cyclic AMP-stimulated phosphorylation of a protein in Limulus ventral and lateral eyes

Abstract

The biogenic amine octopamine (OCT) fulfills most of the criteria as a neurotransmitter of efferent fibers that project to lateral and ventral eyes of the horseshoe crab, Limulus polyphemus. OCT is synthesized by and released from the efferent fibers, and OCT mimics many of the effects of endogenous efferent activity. OCT stimulates an increase in intracellular adenosine 3′,5′-monophosphate (cAMP) in both ventral and lateral eyes, and many of the physiological effects of OCT in these eyes appear to be mediated via cAMP-dependent mechanisms. Here we show that OCT, acting apparently through an OCT-specific receptor, stimulates the increased phosphorylation of a protein with an apparent molecular weight of 122 kDa in both ventral and lateral eyes. This protein is also phosphorylated in response to 8-bromo cAMP and forskolin, suggesting that its phosphorylation involves activation of a cAMP-dependent protein kinase. We present evidence that the 122 kDa protein may be widely distributed in the Limulus visual system but that its phosphorylation in intact tissue in response to OCT, or agents acting through cAMP, may be restricted to portions containing photoreceptor cell bodies. The 122 kDa protein is quantitatively a major cellular protein in the photoreceptor cell body enriched portions of the ventral eye, its isoelectric point is between pH 6.2 and 6.4, and it is associated with both cell membranes and the cytoplasm. The function of this protein is not yet known. It may be important in mediating one or more of the effects of octopamine on Limulus vision.

A Myosin III fromLimulusEyes Is a Clock-Regulated Phosphoprotein

“…Lateral optic nerves were homogenized in 3-[ Nmorpholino]propanesulfonic acid (MOPS) homogenization buffer with protease inhibitors (Edwards and Battelle, 1987) and centrif uged for 30 min at 130,000 ϫ g in an airf uge (Beckman Instruments, Fullerton, CA). The supernatant was mixed with 0.25 volume of fresh 4ϫ SDS sample buffer (Laemmli, 1970) without bromophenol blue dye.…”

“…The supernatant was mixed with 0.25 volume of fresh 4ϫ SDS sample buffer (Laemmli, 1970) without bromophenol blue dye. The proteins were separated by SDS-PAGE on 7.5% gels (Edwards and Battelle, 1987) and blotted to nitrocellulose (MSI, Westboro, M A) (Towbin et al, 1979) in buffer containing 15% methanol and 0.005% ultrapure SDS. The blot was stained with Ponceau S (Aebershold et al, 1987), and the 122 kDa band was cut out.…”

“…A major biochemical consequence of activating clock input to the lateral eyes in vivo and of elevating cAMP in lateral eyes or ventral photoreceptors in vitro is enhanced phosphorylation of an abundant, soluble visual system-specific protein that has an apparent molecular mass on SDS gels of 122 kDa (pp122) (Edwards and Battelle, 1987;Edwards et al, 1990). We have cloned and sequenced cDNA encoding pp122 from a lateral eye cDNA library and report here that it encodes a myosin III.…”

Opsin1-2, Gqα and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock

“…The other three were incubated in the dark for 4h at room temperature in one of three conditions as detailed in the Materials and methods: (1) OCM, (2) OCM plus OA and IBMX, and (3) OCM plus forskolin and IBMX. In the presence of IBMX, OA and forskolin dramatically elevate cAMP in LEs (Edwards and Battelle, 1987;Dalal and Battelle, 2010). At the end of the incubation period, LE slices were fixed in the dark, and RhOps1-2, RhOps5, RhG q α and RhArr levels were quantified in each slice.…”

“…NINAC motor activity has not been demonstrated; therefore, it has been proposed that G q α translocation involves reversible associations of G q α with the cytoplasmic and rhabdomeral isoforms of NINAC (Montell, 2012). Limulus eyes express a single homolog of NINAC that is distributed throughout the photoreceptor, concentrated at the rhabdoms (Battelle et al, 2001), and is a major target for phosphorylation by the circadian clock via activation of cAMP-dependent protein kinase (Edwards and Battelle, 1987;Battelle et al, 1998;Kempler et al, 2007;Cardasis et al, 2007). Limulus myosin III is not a molecular motor ; however, based on the findings in Drosophila and the results presented here, it is interesting to speculate that the clock-and cAMP-dependent phosphorylation of Limulus myosin III plays a role in the clock-dependent translocation of G q α to the rhabdom.…”