By using PYK2-N as bait in a yeast two-hybrid screen, we have isolated a new family of PYK2-binding proteins designated Nir1, Nir2, and Nir3. The Nir family of proteins shows sequence similarity to the Drosophila
rdgB protein, a protein implicated in the phototransduction signaling pathway (24
). It has been shown that rdgB
mutant flies exhibit light-enhanced retinal degeneration and electroretinogram defects. It was proposed that inactivation of rdgB causes abnormalities in phototransduction in Drosophila
because of aberrations in Ca2+
signaling and/or phosphoinositide metabolism (26
Nirs are multidomain proteins containing six putative transmembrane domains, a calcium-binding region, and a carboxy-terminal domain that functions as a PYK2-binding site. The amino-terminal parts of Nir2 and Nir3 contain an additional PI transfer domain, a similar domain was not found in Nir1. In lysates prepared from brain tissue or cultured cells, Nir proteins form a complex with PYK2 and are tyrosine phosphorylated in response to PYK2 activation. It is now well established that PYK2 is activated by a variety of G-protein agonists such as bradykinin, LPA, and angiotensin II, among many other stimuli (12
). In addition, activation of protein kinase C (PKC) and an increase in the cytosolic calcium concentration lead to the activation of PYK2 (19
). Here we demonstrate that PYK2 forms a complex with Nir proteins leading to their tyrosine phosphorylation both in vitro and in living cells. It is therefore likely that these proteins function as downstream targets of PYK2 in different tissues and cell types. However, other possibilities are that Nir proteins act upstream of PYK2 and that activation of PYK2 mediated by Nirs induces tyrosine phosphorylation of Nir proteins.
Although Nir proteins exhibit different tissue expression patterns (Fig. C), all three proteins are expressed in the brain. We demonstrated that the PYK2 and Nir proteins exhibit similar expression patterns in some, but not all, neuronal cell populations in the rat brain. Expression of PYK2 and Nir proteins was detected in the supraoptic nucleus, in the cortex, and in the middle preoptic area, and both proteins are found to be associated in a complex in lysates prepared from these tissues.
Because of the structural similarities between human Nir proteins and the Drosophila
rdgB homologue and because of the extensive genetic and electrophysiological information concerning the role of rdgB in phototransduction, it is possible that Nirs play a similar biological role in the phototransduction pathway in vertebrates. Indeed, it was recently reported that the murine Nir2 gene rescues the phenotype of rdgB
mutant flies (9
), thus demonstrating the Nir and rdgB proteins are functional homologues in vertebrates and Drosophila
, respectively. We therefore compared the cellular distribution of Nirs in the rat retina. The results presented in Fig. demonstrate that Nir proteins and PYK2 exhibit similar expression patterns in different cell types of the retina (13
). PYK2 is highly expressed in the inner nuclear layer and in the ganglion cell layer, and Nir2 immunoreactivity is detected throughout the retina. The immunoreactivity of Nir1 is mostly confined to the outer plexiform layer, to the ganglion cell layer, and to a population of cells in the inner nuclear layer. Nir3 is highly expressed in the inner segment and in the inner and outer plexiform layers. The differences in the expression patterns of the three Nir proteins may reflect differences in their cellular functions. It is not surprising that proteins involved in PI transfer, such as Nir2 and Nir3, are expressed in the inner segment of the retina, potentially participating in the control of membrane turnover, vesicle trafficking, or vesicle fusion. Nir1 does not have a PI transfer domain and therefore may have another regulatory function. An additional possibility is that Nirs play a role in calcium homeostasis, as was previously proposed for the Drosophila
rdgB protein (32
On the basis of genetic, electrophysiological, and pharmacological studies with Drosophila
, it was proposed that rdgB is involved in the pumping of calcium into intracellular stores and perhaps also out of the cell (32
). In the fly retina, light-induced activation of rhodopsin stimulates a G-protein cascade, leading to the activation of phospholipase Cβ. Upon activation, phospholipase Cβ (NorpA) hydrolyzes PI biphosphate to generate diacylglycerol and Ins(1,4,5)P3
. Diacylglycerol then activates PKC (inaC), and Ins(1,4,5)P3
releases calcium from intracellular stores (40
). Genetic and electrophysiological studies with Drosophila
) placed rdgB downstream of NorpA and inaC, suggesting that rdgB uses its PI transfer domain for restoration of Ptd Ins(4,5)P2
, which was utilized during the activation phase of this G-protein-coupled cascade. The experiments described here suggest that PYK2 functions as an additional regulatory component in this process. PYK2 was shown to be activated by a variety of G-protein agonists, such as bradykinin, LPA, and angiotensin II, as well as by many other extracellular stimuli (12
). In addition, phorbol ester-induced activation of PKC and an increase in the cytosolic calcium concentration lead to the activation of PYK2 (19
). Here we demonstrate that PYK2 forms a complex with Nir proteins, leading to tyrosine phosphorylation of these proteins both in vitro and in living cells. A reasonable possibility is that PYK2 and its Drosophila
homologue function as upstream regulators of Nir and rdgB proteins in response to extracellular signals that stimulate G-protein-coupled receptors.
Finally, the finding that the gene for Nir1 is localized to human chromosome 17p13.1 near the marker D17S938 suggests that Nir1 is a candidate gene for inherited retinal diseases. Indeed, the short arm of chromosome 17 has emerged as a hot spot responsible for distinct retinal disorders (17
); LCA, an autosomal recessive disease responsible for congenital blindness (8
); and CORD5, an inherited retinal dystrophy that causes visual impairment and autosomal dominant retinitis pigmentosa. While retinal guanylate cyclase has been shown to be involved in LCA, the gene responsible for CORD5 has not been identified (4
). In addition, the human Nir2-encoding gene was mapped to chromosome 11q13.1 at a locus where genes for several retinal diseases have also been mapped (1
). It remains to be determined whether Nir proteins play a role in these retinal disorders.