Mammalian color vision is mediated by fight-sensitive pigments in retinal cone cells. Biochemical studies on native mammalian cone visual pigments are seriously hampered by their low levels and instability. We describe a novel approach for their functional expression, employing the baculovirus system in combination with histidine tagging to allow future purification and structural analysis. The human red and green cone pigments are produced in relatively large amounts and can be detected by immunocytochemistry as well as by immunoblotting. Histidine tagging has no significant effect on the absorhance maxima. The first evidence is presented that these pigments are N-glycosylated.

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

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