Developmentally regulated alternative splicing of densin modulates protein–protein interaction and subcellular localization

Abstract

Densin is a member of the leucine‐rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C‐terminal domains, including calcium/calmodulin‐dependent protein kinase II (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein–protein interaction domains. For example, CaMKIIα coimmunoprecipitates with densin splice variants containing an intact C‐terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N‐terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N‐ and C‐ terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)‐fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane‐associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.

Characterization of a Central Ca2+/Calmodulin-dependent Protein Kinase IIα/β Binding Domain in Densin That Selectively Modulates Glutamate Receptor Subunit Phosphorylation

“…After blocking in TTBS (50 mM Tris-HCl, pH 7.5, 0.1% (v/v) Tween 20, 150 mM NaCl) containing 5% Carnation nonfat milk, membranes were incubated for either 2 h at room temperature or overnight at 4°C with primary antibodies diluted in TTBS with 5% milk. For densin, antibody R-300 was from Santa Cruz Biotechnology, and Ab450 and Ab650 were described previously (9). Antibodies for total and phospho-Ser-831 GluA1 were from Abcam (Cambridge, MA) and PhosphoSolutions (Aurora, CO), respectively.…”

“…Mammalian Expression Vectors-The expression vector for densin-FLA (densin-⌬1292-1337) with a C-terminal GFP tag was described previously (9). An expression vector for GFPtagged densin-FLC (densin-⌬1292-1337/⌬1378 -1451) was generated similarly in the pEGFP vector (Clontech, BD Biosciences).…”

“…The untagged CaMKII␣ was described previously (9). GFP-CaMKII␤ (WT and T287D) vectors were a generous gift from Dr. Lori Redmond (Medical College of Georgia) (21).…”

“…Initial studies found that the C-terminal association domain of CaMKII␣, but not CaMKII␤, interacts with a CaMKII binding site immediately preceding the C-terminal PDZ domain of densin (6,7), suggesting that densin is a CaMKII␣-specific CaMKAP. Indeed, this C-terminal CaMKII binding site is sufficient to associate with and target CaMKII␣ in intact cells (6,9). However, densin variants are expressed in embryos and in the early postnatal period before expression of CaMKII␣ (9).…”

“…Indeed, this C-terminal CaMKII binding site is sufficient to associate with and target CaMKII␣ in intact cells (6,9). However, densin variants are expressed in embryos and in the early postnatal period before expression of CaMKII␣ (9). Moreover, densin-dependent facilitation of Ca V 1.3 L-type calcium channels by CaMKII␣ is ablated by deletion of a large intracellular domain in densin (⌬483-1377) even though this protein retained the LRR, C-terminal CaMKII␣ binding, and PDZ domains.…”