THE SUBCELLULAR AND CELLULAR DISTRIBUTION OF G PROTEIN-COUPLED RECEPTOR KINASE 2 IN RAT BRAIN C. MURGA, P. PENELA, F. ZAFRA and F. MAYOR JR* Departamento de Biologı ´a Molecular and Centro de Biologı ´a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Universidad Auto ´noma de Madrid, 28049 Madrid, Spain Abstract––G protein-coupled receptor kinase 2 has been found to phosphorylate and thus regulate the activity of several G protein-coupled receptors implicated in neuronal signalling pathways. Although this kinase was initially described as a soluble protein, our laboratory has recently found that a signifi- cant amount of G protein-coupled receptor kinase 2 is associated with microsomal membranes in liver and different types of cultured cells. In the present report we show that high G protein-coupled receptor kinase 2 specific activity and protein levels are present in microsomal fractions of rat brain homogenates. On the other hand, immunochemical detection using a new antibody raised against the N-terminus of the kinase revealed a specific and widely distributed staining in different areas of the central nervous system, and the association of G protein-coupled receptor kinase 2 with intracellular structures in nervous cells. Our results further suggest that this receptor kinase may be involved in the modulation of G protein-coupled receptor-mediated neurotransmission and that association with microsomal membranes may play a role in G protein-coupled receptor kinase 2 functions in the brain.  1998 IBRO. Published by Elsevier Science Ltd. Key words:G protein-coupled receptor kinases (GRK), G protein-coupled receptors (GPCR), -adrenergic receptor kinase (ARK), desensitization, subcellular distribution. G protein-coupled receptors (GPCRs) are a wide- spread family of seven transmembrane domain polypeptides able to transduce extracellular signals by coupling to heterotrimeric G proteins. Numerous members of the GPCR family have been shown to play an important role in synaptic signal transduc- tion, either as neurotransmitter receptors or as sensory targets (light, odorants, etc). 19 The rapid regulation of the functionality of these proteins can be achieved via phosphorylation by a family of specific kinases termed GRKs for G protein-coupled receptor kinases. 9,14 The GRK-phosphorylated receptors are then recognized by proteins of the -arrestin family that promote the termination of the signal. 11,18 Phosphorylation by GRK2 and the sub- sequent -arrestin binding have also been recently implicated in transient receptor internalization from the plasma membrane. 8,21 Of the six GRKs cloned to date, three have been shown to be ubiquitously expressed, namely GRK2, 3 and 5. In particular, GRK2 (also named ARK1 for -adrenergic receptor kinase) is most abundantly expressed in the brain and in peripheral blood cells. 3,5 GRK2 has been reported to specifically phosphor- ylate the agonist-bound form of an increasing number of GPCRs present in the nervous tissue such as adrenergic, muscarinic, angiotensin and substance P receptors, among others. 9,18 However, very little is known about its cellular and subcellular localization in the CNS. Using in situ hybridization techniques and immunoelectron microscopy approaches, one study 1 has described that GRK2 is present in high amounts in a wide variety of brain structures and also that this is the most abundantly expressed GRK isoform in the brain. However, the characteriz- ation of the cellular and subcellular distribution of GRK2 was not addressed in detail. In this regard, recent work from our laboratory has unveiled a rather complex subcellular distribution for GRK2. Apart from being a cytosolic protein transiently translocating to the plasma membrane upon receptor stimulation, GRK2 is also largely associated with internal microsomal membranes in rat liver and several cell lines. 7 We therefore sought to investigate the pattern of GRK2 subcellular distribution in brain both by analysing its activity and levels in different fractions of forebrain homogenates and by immunochemical staining using a new polyclonal antibody raised against the N-terminal region of GRK2. *To whom correspondence should be addressed. A bbreviations: ARK, -adrenergic receptor kinase; BSA, bovine serum albumin; EDTA, ethylenediaminetetra- acetate; EGTA, ethyleneglycolbis(aminoethyl)tetra- acetate; GPCR, G protein-coupled receptors; GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase; SDS–PAGE, sodium dodecyl sulphate– polyacrylamide gel electrophoresis. Pergamon N euroscience Vol. 87, No. 3, pp. 631–637, 1998 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+ 0.00 PII: S0306-4522(98)00145-6 631