Lowell G. Sheflin

Effect of G Protein-Coupled Receptor Kinase 1 (Grk1) Overexpression on Rod Photoreceptor Cell Viability

PURPOSE Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death. METHODS Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light. RESULTS Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times. CONCLUSIONS Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: Phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.

Hormonal Regulation of the EGF/Receptor System

The EGF/receptor system is involved in the development, maintenance and repair of various organs, particularly those involving ductal systems. Disruptions of the EGF/receptor system have been implicated in a variety of clinical disorders including cancer and aging. Some hormones alter the expression of EGF ligands, EGF receptors and of downstream factors that mediate EGF receptor action during development. These hormonal effects vary according to the species, organ and stage of development under study. Androgens alter EGF mRNA levels in several rodent tissues, yet no androgen-responsiveness is detectable within 7 kB upstream of the promoter of the EGF gene, which supports earlier evidence that androgens have post-transcriptional actions on EGF expression. AU-rich elements that can influence transcript stability and subcellular localization are present in the 3’ UTR of EGF mRNA. Androgens regulate EGF mRNA polyadenylation site usage, poly-A tail length and mRNA stability in the mouse submaxillary salivary gland (SMG) but not in the kidney, although androgens do regulate other genes in the kidney. Androgens also regulate the levels of several AU-rich RNA binding proteins known to affect the subcellular localization, translation and stability of AU-rich mRNAs. Androgen-dependent changes in the subcellular levels of these RNA binding proteins also indicate a causal connection with the androgen-dependent changes in EGF mRNA and its translation.