G protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells

• Published in: The Journal of biological chemistry 1997-04, Vol.272 (15), p.10188-10195
• Main Authors: Sallese, M, Mariggiò, S, Collodel, G, Moretti, E, Piomboni, P, Baccetti, B, De Blasi, A
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calmodulin - metabolism; G-Protein-Coupled Receptor Kinase 4; Humans; Isoenzymes - chemistry; Isoenzymes - genetics; Male; Mice; Microscopy, Electron; Molecular Sequence Data; Protein-Serine-Threonine Kinases; Rats; Receptor Protein-Tyrosine Kinases - chemistry; Receptor Protein-Tyrosine Kinases - genetics; RNA Splicing; RNA, Messenger - metabolism; Spermatozoa - enzymology; Spermatozoa - ultrastructure; Testis - enzymology; Testis - ultrastructure
• ISSN: 0021-9258
• DOI: 10.1074/jbc.272.15.10188

G protein-coupled receptor kinase 4 (GRK4) presents some peculiar characteristics that make it a unique member within the GRK multigene family. For example, this is the only GRK for which four splice variants (GRK4alpha, -beta, -gamma, -delta) have been identified. We developed a simple assay to study kinase activity, and we found that GRK4alpha, but not GRK4beta, -gamma, and -delta, was able to phosphorylate rhodopsin in an agonist-dependent manner. GRK4alpha kinase activity was inhibited by Ca2+/calmodulin (CaM) (IC50 = 80 nM), and a direct interaction between GRK4alpha and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The other three GRK4 isoforms did not interact with CaM in parallel experiments. The present investigation also aimed to define cellular and ultrastructural localization of GRK4. A substantial expression of GRK4 mRNA was only found in testis and in the spermatogonia cell line GC-1 spg. Specific GRK4 immunoreactivity was only found on sperm membranes, and immunochemical and ultrastructural analyses showed that it is associated to the acrosomal membranes and to the outer mitochondrial membranes. GRK4gamma was the only detectable isoform in human sperm. We concluded that: i) only GRK4alpha can phosphorylate rhodopsin and that this activity is inhibited by CaM; ii) the other three isoforms do not phosphorylate rhodopsin and do not interact with CaM; and iii) the association of GRK4 with highly specialized sperm organelles, which are essential for fertilization, strongly indicates that this kinase is involved in this process.